Malate dehyrogenases

ABSTRACT

The present invention relates to a recombinant host cell which is capable of producing a dicarboxylic acid and which comprises a mutant malate dehydrogenase resulting in an increased production of the dicarboxylic acid. The invention also relates to a process for producing a dicarboxylic acid, which method comprises fermenting said recombinant host cell in a suitable fermentation medium and producing the dicarboxylic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.16/316,988, filed 10 Jan. 2019, which is a National Stage entry ofInternational Application No. PCT/EP2017/067318, filed 11 Jul. 2017,which claims priority to European Patent Application No. 16179315.3,filed 13 Jul. 2016. Each of these applications is incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2919208-497001_Sequence_Listing_ST25.txt” createdon 12 Aug. 2020, and 143,341 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a recombinant host cell capable ofproducing a dicarboxylic acid, and a method for producing a dicarboxylicacid using said recombinant host cell.

BACKGROUND TO THE INVENTION

The 4-carbon dicarboxylic acids malic acid, fumaric acid and succinicacid are potential precursors for numerous chemicals. For example,succinic acid can be converted into 1,4-butanediol (BDO),tetrahydrofuran, and gamma-butyrolactone. Another product derived fromsuccinic acid is a polyester polymer which is made by linking succinicacid and BDO.

Succinic acid for industrial use is predominantly petrochemicallyproduced from butane through catalytic hydrogenation of maleic acid ormaleic anhydride. These processes are considered harmful for theenvironment and costly. The fermentative production of succinic acid isconsidered an attractive alternative process for the production ofsuccinic acid, wherein renewable feedstock as a carbon source may beused.

Several studies have been carried out on the fermentative production ofC4-dicarboxylic acid in (recombinant) yeast.

EP2495304, for example, discloses a recombinant yeast suitable forsuccinic acid production, genetically modified with genes encoding apyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a malatedehydrogenase, a fumarase, a fumarate reductase and a succinatetransporter.

Despite the improvements that have been made in the fermentativeproduction of dicarboxylic acid in host cells, such as yeast, therenevertheless remains a need for further improved host cells for thefermentative production of dicarboxylic acids.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant host cell which iscapable of producing a dicarboxylic acid and which comprises a mutantpolypeptide having malate dehydrogenase (MDH) activity. Surprisingly, itwas found that the host cell according to the present invention producesan increased amount of a dicarboxylic acid as compared to the amount ofdicarboxylic acid produced by a host cell comprising a reference MDHpolypeptide, the reference MDH polypeptide being typically aNAD(H)-dependent malate dehydrogenase (EC 1.1.1.37).

According to the present invention, there is thus provided a recombinanthost cell which is capable of producing a dicarboxylic acid and whichcomprises a nucleic acid sequence encoding a mutant polypeptide havingmalate dehydrogenase activity, wherein the mutant polypeptide comprisesan amino acid sequence which, when aligned with the malate dehydrogenasecomprising the sequence set out in SEQ ID NO: 39, comprises one mutation(e.g. one substitution) of an amino acid residue corresponding to aminoacid 34 in SEQ ID NO: 39. Said mutant polypeptide having malatedehydrogenase activity may further comprise one or more additionalmutations (e.g. substitutions). In particular, the mutant polypeptidehaving malate dehydrogenase activity may further comprise one or moreadditional mutations (e.g. substitutions) corresponding to any of aminoacids 35, 36, 37, 38, 39 and/or 40 in SEQ ID NO: 39.

According to the present invention, there is also provided a recombinanthost cell which is capable of producing a dicarboxylic acid and whichcomprises a nucleic acid sequence encoding a mutant polypeptide havingmalate dehydrogenase activity, wherein the mutant polypeptide has anincrease in the NADP(H)-relative to NAD(H)-dependent activity ascompared to that of a reference MDH polypeptide, the reference MDHpolypeptide being typically a NAD(H)-dependent malate dehydrogenase (EC1.1.1.37). In said embodiment, said mutant polypeptide may be a mutantpolypeptide comprising an amino acid sequence which, when aligned withthe malate dehydrogenase comprising the sequence set out in SEQ ID NO:39, comprises one mutation (e.g. one substitution) of an amino acidresidue corresponding to amino acid 34 in SEQ ID NO: 39.

The invention also provides:

-   -   a recombinant host cell according to the present invention,        wherein the nucleic sequence encoding the mutant polypeptide        having malate dehydrogenase activity is expressed in the cytosol        and the mutant polypeptide having malate dehydrogenase activity        is active in the cytosol.    -   a recombinant host cell according to the present invention,        wherein the recombinant host cell has an active reductive        tricarboxylic acid (TCA) pathway from phosphoenol or pyruvate to        succinate.    -   a method for the production of a dicarboxylic acid, wherein the        method comprises fermenting the recombinant host cell of the        present invention under conditions suitable for production of        the dicarboxylic acid. The dicarboxylic acid may be succinic        acid, malic acid and/or fumaric acid. The method may further        comprise recovering the dicarboxylic acid from the fermentation        medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out a schematic depiction of integration of fragments 9 to12. The hatched parts indicated in fragments 9 to 12 denote the uniquehomologous overlap regions leading to the recombination events asindicated by the dashed crosses between the homologous regions. The 5′end of fragment 9 and the 3′ end of fragment 12 (indicated by the greyregions in fragments 9 and 12) are homologous to the YPRCtau3 locus onchromosome 16. Homologous recombination results in integration offragment 10 and 11 into the YPRCtau3 locus.

FIG. 2 sets out a schematic depiction of integration of fragments 1-8and fragment 113. The hatched parts indicated in fragments 1-8 and 113denote the unique homologous overlap regions leading to therecombination events as indicated by the dashed crosses between thehomologous regions. Fragment 1 and fragment 113 are homologous to theINT59 locus on chromosome XI, homologous recombination results inintegration of fragment 2-8 into the INT59 locus.

FIG. 3 sets out a schematic depiction of integration of fragments 13,114, 115, 15 and 16. The hatched parts indicated in fragments 13, 114,115, 15 and 16 denote the unique homologous overlap regions leading tothe recombination events as indicated by the dashed crosses between thehomologous regions. Fragment 13 and fragment 16 are homologous to theINT1 locus on chromosome XV, homologous recombination results inintegration of fragment 114, 115 and 15 into the INT1 locus.

FIG. 4 sets out a schematic depiction of integration of fragments 13,14, 16 and one of the fragments 17-110. The hatched parts indicated infragments 13, 14, 16 and fragment 17-110 denote the unique homologousoverlap regions leading to the recombination events as indicated by thedashed crosses between the homologous regions. Fragment 13 and fragment16 are homologous to the INT1 locus on chromosome XV, homologousrecombination results in integration of fragment 14 and one of thefragments 17-112 into the INT1 locus.

FIG. 5A: Average malic acid titers measured in the supernatant ofproduction medium after cultivation of SUC-1112 transformants,expressing phosphoenolpyruvate carboxykinase (PCKa), pyruvatecarboxylase (PYC2), malate dehydrogenase (MDH3), fumarase (FUMR andfumB), dicarboxylic acid transporter (DCT_02) and transformed withreference malate dehydrogenase (SEQ ID NO: 39) or mutant malatedehydrogenase, which contains mutations as compared to the referencesequence in the amino acid positions as indicated in Table 1. The malicacid titer was measured as described in General Materials and Methodsand represents an average value obtained from three independent clones.

FIG. 5B: NADH-specific malate dehydrogenase (MDH) activity of MDHmutants expressed in strain SUC-1112 (see Table 1 for specificmutations). Shown is activity, depicted as Δ A340/min/mg total protein.The value is negative as MDH-dependent NADH oxidation results in adecrease in absorbance at 340 nm. A more negative value indicates moreactivity. The activity was measured as described in Example 5.

FIG. 5C: NADPH-specific malate dehydrogenase (MDH) activity of MDHmutants expressed in strain SUC-1112 (see Table 1 for specificmutations). Shown is activity, depicted as Δ A340/min/mg total protein.The value is negative as MDH-dependent NADPH oxidation results in adecrease in absorbance at 340 nm. A more negative value indicates moreactivity. The activity was measured as described in Example 5.

FIG. 5D: Ratio of NADPH:NADH dependent activity of MDH mutants expressedin strain SUC-1112 (see Table 1 for specific mutations). The activitywas measured and the ratio was determined as described in Example 5. Thedashed line indicates a NADPH:NADH ratio of 1.0.

FIG. 6 sets out a schematic depiction of integration of fragments 116,117, 118 and fragment 119. The hatched parts indicated in fragments 116,117, 118 and fragment 119 denote the unique homologous overlap regionsleading to the recombination events as indicated by the dashed crossesbetween the homologous regions. Fragment 116 and fragment 119 arehomologous to the INT1 locus on chromosome XV, homologous recombinationresults in integration of fragment 117 and fragment 118 into the INT1locus.

FIG. 7 sets out a schematic depiction of integration of fragments 1-5,124 and fragment 120, 121, 122 or 123. The hatched parts indicated inthe fragments denote the unique homologous overlap regions leading tothe recombination events as indicated by the dashed crosses between thehomologous regions. Fragment 1 and fragment 124 are homologous to theINT59 locus on chromosome XI, homologous recombination results inintegration of fragment 2-5 and 120, 121, 122 or 123 into the INT59locus.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the nucleotide sequence of fragment 2 (FIG. 2),which includes PEP carboxykinase from Actinobacillus succinogenes codonpair optimized for expression in Saccharomyces cerevisiae.

SEQ ID NO: 2 sets out the nucleotide sequence of fragment 3 (FIG. 2),which includes pyruvate carboxylase (PYC2) from S. cerevisiae codon pairoptimized for expression in S. cerevisiae.

SEQ ID NO: 3 sets out the nucleotide sequence of the PCR template forfragment 4 (FIG. 2), which includes a KanMX selection marker functionalin S. cerevisiae.

SEQ ID NO: 4 sets out the nucleotide sequence of fragment 5 (FIG. 2),which includes a putative dicarboxylic acid transporter from Aspergillusniger codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 5 sets out the nucleotide sequence of fragment 6 (FIG. 2),which includes malate dehydrogenase (MDH3) from S. cerevisiae codon pairoptimized for expression in S. cerevisiae.

SEQ ID NO: 6 sets out the nucleotide sequence of fragment 7 (FIG. 2),which includes fumarase (fumB) from Escherichia coli codon pairoptimized for expression in S. cerevisiae.

SEQ ID NO: 7 sets out the nucleotide sequence of fragment 8 (FIG. 2),which includes fumarate reductase from Trypanosoma brucei (FRDg) codonpair optimized for expression in S. cerevisiae.

SEQ ID NO: 8 sets out the amino acid sequence of fumarate reductase fromTrypanosoma brucei (FRDg).

SEQ ID NO: 9 sets out the nucleotide sequence of the primer used togenerate fragment 1 (FIG. 2).

SEQ ID NO: 10 sets out the nucleotide sequence of the primer used togenerate fragment 1 (FIG. 2).

SEQ ID NO: 11 sets out the nucleotide sequence of the primer used togenerate fragment 2 (FIG. 2).

SEQ ID NO: 12 sets out the nucleotide sequence of the primer used togenerate fragment 2 (FIG. 2).

SEQ ID NO: 13 sets out the nucleotide sequence of the primer used togenerate fragment 3 (FIG. 2).

SEQ ID NO: 14 sets out the nucleotide sequence of the primer used togenerate fragment 3 (FIG. 2).

SEQ ID NO: 15 sets out the nucleotide sequence of the primer used togenerate fragment 4 (FIG. 2).

SEQ ID NO: 16 sets out the nucleotide sequence of the primer used togenerate fragment 4 (FIG. 2).

SEQ ID NO: 17 sets out the nucleotide sequence of the primer used togenerate fragment 5 (FIG. 2).

SEQ ID NO: 18 sets out the nucleotide sequence of the primer used togenerate fragment 5 (FIG. 2).

SEQ ID NO: 19 sets out the nucleotide sequence of the primer used togenerate fragment 6 (FIG. 2) and fragments 120, 121, 122 and 123 (FIG.7).

SEQ ID NO: 20 sets out the nucleotide sequence of the primer used togenerate fragment 6 (FIG. 2) and fragments 120, 121, 122 and 123 (FIG.7).

SEQ ID NO: 21 sets out the nucleotide sequence of the primer used togenerate fragment 7 (FIG. 2).

SEQ ID NO: 22 sets out the nucleotide sequence of the primer used togenerate fragment 7 (FIG. 2).

SEQ ID NO: 23 sets out the nucleotide sequence of the primer used togenerate fragment 8 (FIG. 2).

SEQ ID NO: 24 sets out the nucleotide sequence of the primer used togenerate fragment 8 (FIG. 2).

SEQ ID NO: 25 sets out the nucleotide sequence of the primer used togenerate fragment 13 (FIG. 3).

SEQ ID NO: 26 sets out the nucleotide sequence of the primer used togenerate fragment 13 (FIG. 3).

SEQ ID NO: 27 sets out the nucleotide sequence of the primer used togenerate fragment 14 (FIG. 4).

SEQ ID NO: 28 sets out the nucleotide sequence of the primer used togenerate fragment 115 (FIG. 3) and fragment 14 (FIG. 4).

SEQ ID NO: 29 sets out the nucleotide sequence of the primer used togenerate fragment 15 (FIG. 3) and fragments 17 to 110 (FIG. 4).

SEQ ID NO: 30 sets out the nucleotide sequence of the primer used togenerate fragment 15 (FIG. 3) and fragments 17 to 110 (FIG. 4).

SEQ ID NO: 31 sets out the nucleotide sequence of fragment 15 (FIG. 3)and fragment 120 (FIG. 7), which includes the nucleotide sequenceencoding SEQ ID NO: 39 codon pair optimized for expression in S.cerevisiae.

SEQ ID NO: 32 sets out the nucleotide sequence of the primer used togenerate fragment 16 (FIG. 3).

SEQ ID NO: 33 sets out the nucleotide sequence of the primer used togenerate fragment 16 (FIG. 3).

SEQ ID NO: 34 sets out the nucleotide sequence of fragment 9 (FIG. 1),which includes fumarase from Rhizopus oryzae codon pair optimized forexpression in Saccharomyces cerevisiae.

SEQ ID NO: 35 sets out the nucleotide sequence of fragment 10 (FIG. 1),which includes the 5′ part of the Cre-recombinase.

SEQ ID NO: 36 sets out the nucleotide sequence of fragment 11 (FIG. 1),which includes the 3′ part of the Cre-recombinase.

SEQ ID NO: 37 sets out the nucleotide sequence of fragment 12 (FIG. 1),which includes a region homologous to the YPRCtau3 locus.

SEQ ID NO: 38 sets out the nucleotide sequence of the PCR template forfragment 115 (FIG. 3), fragment 14 (FIG. 4) and fragment 117 (FIG. 6),which includes the nourseothricin selection marker.

SEQ ID NO: 39 sets out the amino acid sequence of the malatedehydrogenase (MDH3) protein from S. cerevisiae, lacking the 3C-terminal peroxisomal targeting sequence.

SEQ ID NO: 40 sets out the nucleotide sequence of the primer used togenerate fragment 113 (FIG. 2).

SEQ ID NO: 41 sets out the nucleotide sequence of the primer used togenerate fragment 113 (FIG. 2).

SEQ ID NO: 42 sets out the nucleotide sequence of fragment 114 (FIG. 3),which includes the expression cassette of ZWF1 from S. cerevisiae codonpair optimized for expression in S. cerevisiae.

SEQ ID NO: 43 sets out the nucleotide sequence of the primer used togenerate fragment 114 (FIG. 3).

SEQ ID NO: 44 sets out the nucleotide sequence of the primer used togenerate fragment 114 (FIG. 3).

SEQ ID NO: 45 sets out the nucleotide sequence of the primer used togenerate fragment 115 (FIG. 3).

SEQ ID NO: 46 sets out the amino acid sequence of the pyruvatecarboxylase protein from S. cerevisiae.

SEQ ID NO: 47 sets out the amino acid sequence of phosphoenolpyruvatecarboxykinase from Actinobacillus succinogenes, with EGY to DAFmodification at position 120-122.

SEQ ID NO: 48 sets out the amino acid sequence of fumarase (fumB) fromEscherichia coli.

SEQ ID NO: 49 sets out the amino acid sequence of fumarase from Rhizopusoryzae, lacking the first 23 N-terminal amino acids.

SEQ ID NO: 50 sets out the amino acid sequence of a putativedicarboxylic acid transporter from Aspergillus niger.

SEQ ID NO: 51 sets out the amino acid sequence of isocitrate lyase fromKluyveromyces lactis.

SEQ ID NO: 52 sets out the amino acid sequence of Saccharomycescerevisiae peroxisomal malate synthase (Mls1) amino acid sequence,lacking the 3 C-terminal peroxisomal targeting sequence.

SEQ ID NO: 53 sets out the amino acid sequence of the malatedehydrogenase (MDH3) protein from S. cerevisiae, including theperoxisomal targeting sequence SKL.

SEQ ID NO: 54 sets out the nucleotide sequence of the primer used togenerate fragment 116 (FIG. 6).

SEQ ID NO: 55 sets out the nucleotide sequence of the primer used togenerate fragment 116 (FIG. 6).

SEQ ID NO: 56 sets out the nucleotide sequence of the primer used togenerate fragment 117 (FIG. 6).

SEQ ID NO: 57 sets out the nucleotide sequence of the primer used togenerate fragment 117 (FIG. 6).

SEQ ID NO: 58 sets out the nucleotide sequence of the primer used togenerate fragment 119 (FIG. 6).

SEQ ID NO: 59 sets out the nucleotide sequence of the primer used togenerate fragment 119 (FIG. 6).

SEQ ID NO: 60 sets out the nucleotide sequence of the primer used togenerate fragment 118 (FIG. 6).

SEQ ID NO: 61 sets out the nucleotide sequence of the primer used togenerate fragment 118 (FIG. 6).

SEQ ID NO: 62 sets out the nucleotide sequence of fragment 118 (FIG. 6)which includes coding sequence for fumarate reductase from Trypanosomabrucei (FRDg) codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 63 sets out the nucleotide sequence of the primer used togenerate fragment 124 (FIG. 7).

SEQ ID NO: 64 sets out the nucleotide sequence of fragment 121 (FIG. 7)which includes coding sequence for S. cerevisiae MDH3 mutant MUT_014codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 65 sets out the nucleotide sequence of fragment 122 (FIG. 7)which includes coding sequence for S. cerevisiae MDH3 mutant MUT_015codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 66 sets out the nucleotide sequence of fragment 123 (FIG. 7)which includes coding sequence for S. cerevisiae MDH3 mutant MUT_034codon pair optimized for expression in S. cerevisiae.

SEQ ID NO: 67 sets out the amino acid sequence of fumarase fromArabidopsis thaliana.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims, thewords “comprise”, “include” and “having” and variations such as“comprises”, “comprising”, “includes” and “including” are to beinterpreted inclusively. That is, these words are intended to convey thepossible inclusion of other elements or integers not specificallyrecited, where the context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

The reductive TCA pathway is one of the primary pathways by which amicroorganism can produce dicarboxylic acids. In recent years, it hasproven to be the best economic option for the microbial production ofdicarboxylic acids, e.g. succinic acid. The reductive TCA pathwayincludes two reactions which require the consumption of reducing power;i.e. the malate dehydrogenase reaction (reduction of oxaloacetate tomalate) and the fumarate reductase reaction (reduction of fumarate tosuccinate).

Malate dehydrogenase (MDH) catalyzes the reversible conversion of malateto oxaloacetate using NAD or NADP as the cofactor (also collectivelyreferred as NAD(P)). MDH is a rather ubiquitous enzyme and plays crucialroles in many metabolic pathways, including the tricarboxylic acidcycle, amino acid synthesis, gluconeogenesis, maintenance of theoxidation/reduction balance and metabolic stress.

MDHs can be divided into NAD(H)-dependent MDHs (NAD-MDH) (EC 1.1.1.37)and NADP(H)-dependent MDHs (NADP-MDH) (EC 1.1.1.82), according to theirpreference for cofactors. Most bacterial and archaeal MDHs are NAD-MDHs.Eukaryotic MDH isoforms are all NAD-MDHs, including mitochondrial MDHs,cytosolic MDHs, glyoxysomal MDHs, and peroxisomal MDHs, except forchloroplastic NADP-MDHs, which are required for the transfer of reducingequivalents from chloroplast stroma to cytosol. In the yeastSaccharomyces cerevisiae, three endogenous isoenzymes of malatedehydrogeneases have been identified, namely MDH1, MDH2 and MDH3. Theywere located in the mitochondria (MDH1), the cytosol (MDH2) and theperoxisome (MDH3) and were all characterized to be NAD(H)-dependent MDHs(EC 1.1.1.37).

The study of NAD(P)-binding domains in the malate dehydrogenase enzymefamily revealed a conserved βB-αC motif of the Rossmann fold. Theability of the dehydrogenases to discriminate against NADP(H) lies inthe amino acid sequence of this βB-αC motif, which has been predicted tobe a principal determinant for cofactor specificity. For example, in theS. cerevisiae peroxisomal NAD-MDH (MDH3), the NAD-binding motif includesamino acid residues 34 to 40 which were found important for cofactorbinding and specificity.

In the context of the present invention, it has been surprisingly foundthat a set of specific mutations in the conserved NAD-binding motif of apolypeptide having MDH activity confers an increased production of adicarboxylic acid when (over)expressed in a recombinant host cellcapable of the production of said dicarboxylic acid. That is to say,(over)expression of said mutant polypeptide having MDH activity in arecombinant host cell typically leads to increased production of adicarboxylic acid as compared to a recombinant host cell which(over)expresses a reference MDH polypeptide; the “reference MDHpolypeptide” being typically a NAD-MDH (EC 1.1.1.37). Concomitantly, ithas been shown that said mutant polypeptide having at least one mutationin the conserved NAD-binding motif has an increase in theNADP(H)-relative to NAD(H)-dependent activity as compared to that ofsaid reference MDH polypeptide. Surprisingly, the inventors of thepresent invention have further shown that the NADP(H)-dependent activitydoes not have to be higher than the NAD(H)-dependent activity to obtainan increase in dicarboxylic acid production.

It is therefore an object of the present invention to provide arecombinant host cell which is capable of producing a dicarboxylic acidand which comprises a mutant polypeptide having malate dehydrogenase(MDH) activity.

In one embodiment, the mutant polypeptide having malate dehydrogenaseactivity comprises an amino acid sequence which, when aligned with themalate dehydrogenase comprising the sequence set out in SEQ ID NO: 39,comprises one mutation of an amino acid residue corresponding to aminoacid 34 in SEQ ID NO: 39. In other words, said mutant polypeptidecomprises one mutation of an amino acid residue occurring at a positioncorresponding to 34 in SEQ ID NO: 39.

In a preferred embodiment of the invention, the mutation of the aminoacid corresponding to amino acid 34 (as defined with reference to SEQ IDNO: 39) will be a substitution.

More preferably, the substitution of the amino acid corresponding toamino acid 34 (as defined with reference to SEQ ID NO: 39) willtypically be to a small amino acid. Suitable small amino acids includethreonine (T), serine (S), glycine (G), alanine (A) and proline (P).Preferred small amino acids are glycine (G) and serine (S).

In the context of the present invention, a “recombinant host cell” or a“genetically modified host cell” is a host cell into which has beenintroduced, by means of recombinant DNA techniques, a nucleic acid, anucleic acid construct or a vector comprising a nucleic acid sequenceencoding a mutant polypeptide having malate dehydrogenase activity.

Herein, a “mutant polypeptide having malate dehydrogenase (MDH)activity” may be referred to as a “mutant malate dehydrogenase”, “MDHmutant”, “MDH mutant polypeptide”, “mutant”, “mutant polypeptide” or thelike.

Herein, the “malate dehydrogenase activity” is the activity convertingoxaloacetic acid to malic acid:Malic acid+acceptor<=>oxaloacetic acid+reduced acceptor

The term “polypeptide” is used herein for chains containing more thanabout seven amino acid residues. All polypeptide sequences herein arewritten from left to right and in the direction from amino terminus tocarboxy terminus. The one-letter code of amino acids used herein iscommonly known in the art and can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor Laboratory, N Y, 2001).

In the context of the present invention, a “mutant” polypeptide isdefined as a polypeptide which was obtained by introduction of one ormore mutations. Said mutations may be selected from the group ofsubstitutions, additions and deletions. The term “substitution” hereinmeans the replacement of an amino acid residue in the polypeptidesequence with another one. A “mutant” polypeptide, a “mutated”polypeptide and a “genetically engineered” polypeptide have the samemeaning and are used interchangeably.

Herein, a “corresponding position” refers to the vertical column in anamino acid sequence alignment between SEQ ID NO: 39 and sequenceshomologous to SEQ ID NO: 39 corresponding to a specific position in SEQID NO:39 and showing the amino acids that occur at this position in theother aligned homologues.

In the context of the invention, a “corresponding mutation” refers to amutation of an amino acid residue occurring at a “correspondingposition” in SEQ ID NO: 39. For example, a “corresponding substitution”refers to a substitution of an amino acid residue occurring at a“corresponding position” in SEQ ID NO: 39 with another amino acidresidue.

In some further embodiments, the mutant polypeptide having malatedehydrogenase activity may further comprise one or more additionalmutations corresponding to any of amino acids 35, 36, 37, 38, 39 and/or40 in SEQ ID NO: 39. Said mutations will typically be selected from thegroup of substitutions, additions and deletions. More preferably, theone or more additional mutations will be a substitution.

The substitution of the amino acid corresponding to amino acid 35 (asdefined with reference to SEQ ID NO: 39) will typically be to a smallamino acid. Suitable small amino acids include threonine (T), serine(S), glycine (G), alanine (A) and proline (P). Alternatively, thesubstitution of the amino acid corresponding to amino acid 35 (asdefined with reference to SEQ ID NO: 39) will be to a hydrophobic aminoacid, such as isoleucine (I). A preferred substitution of the amino acidcorresponding to amino acid 35 (as defined with reference to SEQ ID NO:39) will be to serine (S) or isoleucine (I).

The substitution of the amino acid corresponding to amino acid 36 (asdefined with reference to SEQ ID NO: 39) will typically be to a polaramino acid. Suitable polar amino acids include arginine (R), glutamine(Q), Glutamic acid (E) and serine (S). Alternatively, the substitutionof the amino acid corresponding to amino acid 36 (as defined withreference to SEQ ID NO: 39) will be to a small amino acid, such asalanine (A) or proline (P). A preferred substitution of the amino acidcorresponding to amino acid 36 (as defined with reference to SEQ ID NO:39) will be to arginine (R), glutamine (Q), glutamic acid (E), serine(S), alanine (A) or proline (P).

The substitution of the amino acid corresponding to amino acid 37 (asdefined with reference to SEQ ID NO: 39) will typically be to a smallamino acid. Suitable small amino acids include glycine (G), asparagine(N), and alanine (A). Alternatively, the substitution of the amino acidcorresponding to amino acid 37 (as defined with reference to SEQ ID NO:39) will be to a polar amino acid, such as arginine (R) or glutamine(Q). A preferred substitution of the amino acid corresponding to aminoacid 37 (as defined with reference to SEQ ID NO: 39) will be to (G),asparagine (N), alanine (A), alanine arginine (R) or glutamine (Q).

The substitution of the amino acid corresponding to amino acid 38 (asdefined with reference to SEQ ID NO: 39) will typically be to a smallamino acid. Suitable small amino acids include valine (V), threonine(T), serine (S), glycine (G), alanine (A) and proline (P). A preferredsubstitution of the amino acid corresponding to amino acid 38 (asdefined with reference to SEQ ID NO: 39) will be to alanine (A), valine(V), threonine (T) or serine (S).

The substitution of the amino acid corresponding to amino acid 39 (asdefined with reference to SEQ ID NO: 39) will typically be to a smallamino acid. A suitable small amino acid includes proline (P).Alternatively, the substitution of the amino acid corresponding to aminoacid 39 (as defined with reference to SEQ ID NO: 39) will be to ahydrophobic amino acid, such as lysine (K), phenylalanine (F) or leucine(L). Alternatively, the substitution of the amino acid corresponding toamino acid 39 (as defined with reference to SEQ ID NO: 39) will be to apolar amino acid, such as glutamic acid (E). A preferred substitution ofthe amino acid corresponding to amino acid 39 (as defined with referenceto SEQ ID NO: 39) will be to glutamic acid (E), lysine (K),phenylalanine (F), leucine (L) or proline (P).

The substitution of the amino acid corresponding to amino acid 40 (asdefined with reference to SEQ ID NO: 39) will typically be to a smallamino acid. Suitable small amino acids include glycine (G).Alternatively, the substitution of the amino acid corresponding to aminoacid 40 (as defined with reference to SEQ ID NO: 39) will be to a polaramino acid, such as glutamine (Q). A preferred substitution of the aminoacid corresponding to amino acid 40 (as defined with reference to SEQ IDNO: 39) will be to glycine (G) or glutamine (Q).

The various types of amino acids above are classified with reference to,for example, Betts and Russell, In Bioinformatics for Geneticists,Barnes and Gray eds, Wiley 2003.

In more detail, in the context of the invention, a mutant polypeptidehaving malate dehydrogenase activity will comprise G or S at position 34as defined with reference to SEQ ID NO: 39;

and, optionally

I or S at position 35 as defined with reference to SEQ ID NO: 39; and/or

R, Q, A, E, P or S at position 36 as defined with reference to SEQ IDNO: 39; and/or

A, N, G, R or Q at position 37 as defined with reference to SEQ ID NO:39; and/or

A, V, T or S at position 38 as defined with reference to SEQ ID NO: 39;and/or

E, K, P, F or L at position 39 as defined with reference to SEQ ID NO:39; and/or

G or Q at position 40 as defined with reference to SEQ ID NO: 39.

In one specific embodiment, a mutant polypeptide having malatedehydrogenase activity will comprise a small amino acid at position 34(as defined with reference to SEQ ID NO: 39) and a small or polar aminoacid at position 36 (as defined with reference to SEQ ID NO: 39). Insaid embodiment, a preferred small amino acid at position 34 may beselected from G or S. In said embodiment, a preferred small or polaramino acid at position 36 may be selected from R, Q, A, E, P or S.Optionally, in said embodiment, the mutant polypeptide will comprise

I or S at position 35 as defined with reference to SEQ ID NO: 39; and/or

A, N, G, R or Q at position 37 as defined with reference to SEQ ID NO:39; and/or

A, V, T or S at position 38 as defined with reference to SEQ ID NO: 39;and/or

E, K, P, F or L at position 39 as defined with reference to SEQ ID NO:39; and/or

G or Q at position 40 as defined with reference to SEQ ID NO: 39.

The mutant polypeptide having MDH activity may furthermore comprisesadditional mutations other than the seven positions defined above, forexample, one or more additional substitutions, additions or deletions.

The mutant polypeptide having MDH activity may comprise a combination ofdifferent types of modification of this sort. The mutant polypeptidehaving MDH activity may comprise one, two, three, four, least 5, atleast 10, at least 15, at least 20, at least 25, at least 30 or moresuch modifications (which may all be of the same type or may bedifferent types of modification). Typically, the additionalmodifications may be substitutions.

In yet further embodiments, the mutant polypeptide having malatedehydrogenase activity is as defined with reference to Table 1 (Example4) and wherein the amino acid residue corresponding to amino acid 34 inSEQ ID NO: 39 is selected from glycine (G) or serine (S). That is tosay, the mutant polypeptide may comprise any combination ofsubstitutions as set out in Table 1 as compared to a suitable referencesequence such as that set out in SEQ ID NO: 39, and wherein the aminoacid residue corresponding to amino acid 34 in SEQ ID NO: 39 is selectedfrom glycine (G) or serine (5).

Typically, then the mutant polypeptide may comprise the sequence of SEQID NO: 39 with one substitution at position 34, and optionally one ormore substitutions at 35, 36, 37, 38, 39 and/or 40. That is to say, themutant polypeptide will have an amino acid other than aspartate atposition 34 and optionally an amino acid other than isoleucine atposition 35 and/or an amino acid other than arginine at position 36and/or an amino acid other than alanine at position 37, and/or an aminoacid other than alanine at position 38, and/or an amino acid other thanglutamate at position 39, and/or an amino acid other than glycine atposition 40.

Also, typically, the mutant polypeptide may comprise the sequence of SEQID NO: 39 with one substitution at position 34, one substitution atposition 36, and optionally one or more substitutions at 35, 37, 38, 39and/or 40. That is to say, the mutant polypeptide will have an aminoacid other than aspartate at position 34, an amino acid other thanarginine at position 36, and optionally an amino acid other thanisoleucine at position 35 and/or an amino acid other than alanine atposition 37, and/or an amino acid other than alanine at position 38,and/or an amino acid other than glutamate at position 39, and/or anamino acid other than glycine at position 40.

In a separate embodiment of the present invention, the mutantpolypeptide having malate dehydrogenase activity has an increase in theNADP(H)-relative to NAD(H)-dependent activity as compared to that of areference MDH polypeptide. In said embodiment, said mutant polypeptidemay be a mutant polypeptide comprising an amino acid sequence which,when aligned with the malate dehydrogenase comprising the sequence setout in SEQ ID NO: 39, comprises one mutation (e.g. one substitution) ofan amino acid residue corresponding to amino acid 34 in SEQ ID NO: 39.Further embodiments with regard to the amino acid sequence of saidmutant polypeptide are as described herein above.

In the context of the invention, a reference polypeptide having malatedehydrogenase activity, also called a “reference MDH polypeptide”, maybe NAD-MDH (EC 1.1.1.37). A reference polypeptide having malatedehydrogenase activity may be a malate dehydrogenase from a microbialsource, such as a yeast (e.g. Saccharomyces cerevisiae). A malatedehydrogenase having the amino acid sequence set out in SEQ ID NO: 39may be a suitable reference polypeptide having MDH activity.

The expression “increase in NADP(H)-relative to NAD(H)-dependentactivity” herein typically refers to the property of a mutantpolypeptide to show an increase in NADP(H)-relative to NAD(H)-dependentactivity in comparison to that of a reference MDH polypeptide, forexample in comparison to SEQ ID NO: 39. That is to say a mutantpolypeptide may show an increase in the ratio of NADP(H)- toNAD(H)-dependent activity in comparison to that of a referencepolypeptide. In Example 5, the ratio is also referred as the “NADPH:NADHspecificity ratio”.

In the context of the present invention, the terms “NADP(H)-dependentactivity” and “NADP(H)-specific activity” have the same meaning hereinand are used interchangeably. Same applies for the terms“NAD(H)-dependent activity” and “NAD(H)-specific activity”.

The term “NADP(H)-dependent activity” herein refers to the property ofan enzyme to use NADP(H) as the redox cofactor. The NADP(H)-dependentactivity of the enzyme may be determined by an enzyme activity assaysuch as described in Example 5.

The term “NAD(H)-dependent activity” herein refers to the property of anenzyme to use NAD(H) as the redox cofactor. The NAD(H)-dependentactivity of the enzyme may be determined by an enzyme activity assaysuch described in Example 5.

An increased value of the average NADPH:NADH specificity ratio mayindicate, for example, a reduced NAD(H)-dependent activity, an increasedNADP(H)-dependent activity or a combination of the two. In some cases,an increased value of said ratio may be obtained with a MDH mutanthaving a similar or increased NAD(H)-dependent activity in comparison toa reference MDH. In the latter cases, it may be that the MDH mutantdisplays both increased NAD(H)- and NADP(H)-dependent activities.

The mutant MDH polypeptide will typically have modified MDH activity interms of modified cofactor dependence. This NAD(H)- or NADP(H)-dependentactivity may be modified independent from each other, for exampledecreased, by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or at least 99%. Alternatively, the property may be increasedby at least 10%, at least 25%, at least 50%, at least 70%, at least100%, at least, 200%, at least 500%, at least 700%, at least 1000%, atleast 3000%, at least 5000%, or at least 6000%.

In one specific embodiment, the NAD(H)- and NADP(H)-dependent activitiesof the mutant MDH polypeptide are both increased. The NAD(H)-dependentactivity of said mutant MDH polypeptide is increased by at least 10%, atleast 25%, at least 50%, at least 70%, at least 100%, at least, 200%, orat least 300%. The NADP(H)-dependent activity of said mutant MDHpolypeptide is increased by at least 10%, at least 25%, at least 50%, atleast 70%, at least 100%, at least, 200%, at least 500%, at least 700%,at least 1000%, at least 3000%, at least 5000%, or at least 6000%.

In another embodiment, the NAD(H)-dependent activity of the mutant MDHpolypeptide is about the same or decreased by at most 5%, at most 10%,at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most70%, at most 80% and the NADP(H)-dependent activity of said mutant MDHpolypeptide is increased by at least 10%, at least 25%, at least 50%, atleast 70%, at least 100%, at least, 200%, at least 500%, at least 700%,at least 1000%, at least 3000%, at least 5000%, or at least 6000%.

In another embodiment, the NADPH:NADH specificity ratio of the mutantMDH polypeptide is increased by at least 10%, at least 50%, at least100%, at least, 200%, at least 500%, at least 1000%, at least 3000%, atleast 5000%, at least 7000%, or at least 8000%.

The percentage decrease or increase in this context represents thepercentage decrease or increase in comparison to the reference MDHpolypeptide, for example that of SEQ ID NO: 39. It is well known to theskilled person how such percentage changes may be measured—it is acomparison of the activity, for example NAD(H)- or NADP(H)-dependentactivity, of the reference MDH and the mutant MDH measured as set out inthe Examples.

In the context of the present invention, the MDH mutant polypeptide asdescribed herein above may be a mutant NAD(P)-malate dehydrogenase, suchas a mutant mitochondrial NAD-MDH, a mutant cytosolic NAD-MDH, a mutantglyoxysomal NAD-MDH, a mutant peroxisomal NAD-MDH, or a mutantchloroplastic NADP-MDH. That is to say, the mutant polypeptide havingmalate dehydrogenase activity may be obtained by introduction of one ormore mutations in a NAD(P)-malate dehydrogenase, such as a mitochondrialNAD-MDH, cytosolic NAD-MDH, glyoxysomal NAD-MDH, peroxisomal NAD-MDH, ora chloroplastic NADP-MDH (the latter being referred as template MDHpolypeptides for introducing said one or more mutations). Preferably,the mutant polypeptide having malate dehydrogenase activity is a mutantNAD(H)-malate dehydrogenase (EC 1.1.1.37). More preferably, the mutantpolypeptide having malate dehydrogenase activity is a mutant peroxisomalNAD(H)-malate dehydrogenase. Even more preferably, the mutantpolypeptide having malate dehydrogenase activity is a mutantNAD(H)-malate dehydrogenase from a yeast or a fungus. Even morepreferably, the mutant polypeptide having malate dehydrogenase activityis a mutant NAD(H)-malate dehydrogenase from a yeast or fungus, such asS. cerevisiae, Torulaspora delbrueckii, Zygosaccharomyces bailiff,Naumovozyma casteffii, Naumovozyma dairenensis, Lachancea lanzarotensis,Zygosaccharomyces rouxii, Kazachstania africana, Candida tropicalis,Kluyveromyces marxianus, Scheffersomyces stipites, Talaromyces mameffei,Rasamsonia emersonii, Aspergillus niger, or Trametes versicolor. Thefollowing Uniprot database codes refer to suitable yeast and fungaltemplate MDH polypeptides (http://www.uniprot.org): E7NGH7, G8ZXS3,G0V668, W0VUI8, G0WB63, A0A0W0D4X6, A0A0C7MME9, C5DQ42, C5D145, H2AWW6,A0A090C493, J7R0C8, Q6CJP3, Q759M4, I2H037, C5M546, A7TL95, A0A0L0P3G3,Q6BM17, S8AW17, V5FMV2, A3LW84, A0A109UZS1, G8BVW8, G8BJ12, M3HPK4,A0A093UW53, B8MTP0, A0A0F4YPR0, C8V0H6, W6QNU3, A5DZ33, U1GAT6, G3B7S5,C4JP17, A0A0F8UZY9, Q4WDM0, A0A093UPX3, B8ND04, A0A0M9VRI4, G7XZ98,Q5A5S6, M7S9E4, E4UYX5, A5DE02, A0A0J7B1J5, A0A017SKI1, G8Y7A1,A0A0G2EFQ2, R7S165, I1RFM4, R1EVC8, U4L6K9, A0A0L0P507, W7HM94, A5DGY9,F2QY33, A0A0G2JA24, UPI000462180C, C7Z9W6, E5AAQ2, B2VVR8, A0A0H2RCV1,A8Q524, A0A0E9N879, N1JA02, A0A0D1ZEE3, J5T1X5, W6MY07, C4Y826, G3AJA2,G9N6G5, AOAOK8L6L9, A3GH28, A8P7W6, K5W0T4, G1XT67, A0A0B7K175, B8MTP5,A0A0D1ZAS7, A0A0C3BQC4, K5Y2Q9, A0A0C3DSW4, A0A068S518, W2RPL2,A0A0H5C453, A0A074WKG5, G8JRX4, A0A0U1M134, H6C0V9, A0A0H5BZ30, M2UXR7,A0A0C9YJV6, A0A0C3S6T1, W1Q K02, A0A0C2YFC4, A0A061AJ54, A0A086T183,W2RVT1, UPI0004623914, A0A0C9X7U1, UPI0001F26169, G7E054, A0A0C2S9T3,A0A067NIX1, L8FPM0, G3ALW4, A0A0D0AYX2, G0VJG3, A0A0D2NGY0, C1GLB8,W9X415, A0A0D0CDE8, S7Q8G0, A0A0C9TB51, R7YP89, A0A0C2W4H8,UPI000455FA04, A0A067NL73, A0A067STP4, W9VWVP5, A0A0D7A9T7, A0A0D6R2E1,M2YLX9, G0W7D4, N1QJ61, G4TRY5, FOXJ10, A0A063BQQ6, A0A061HBX5,A0A0A1PCT2, M1WIC4, A8QAQ2, A0A0C3N631, F2QTL7, A0A060S7U3, A0A0L0HTQ9,Q0CKY1, A0A0C2ZJ90, K5W527, I4Y5C3, R7YXZ2, F9XI12, A0A061J968, F9XL74,A0A0D0BEW9, A0A0C9WB72, F7WA21, A8NJ67, M2P8M6, W4KHW3, A0A0L1I2T4,UPI0004449A9D, P83778, C4Y9Q7, A0A0D7BHV7, A0A068RWX9, M2YWQ3,A0A137QV51, A0A0D0B6C2, I1BQQ7, S3DA07, Q4DXL5, G8Y022, A0A0C9W9B3,A0A0L6WJ49, A0A0L9SL52, D5GA85, A0A0N1J4Z6, J7S1G2, A0A0F4X5C6, Q6CIK3,A0A067QNN0, Q0UGT7, F8ND69, U5HIM0, J3NKC7, A0A061ATZ7, A5DSY0, Q9Y750,UPI0004F4119A, A0A086TL69, A0A0J0XSS4, UPI0003F496E1, UPI000455F0EA,S7RXX1, A0A067NAG9, A0A0B7N3M5, E6ZKH0, C8V1V3, A7UFI6, T5AEM1,A0A072PGA9, A0A094EKH6, S8ADX4, G8C073, Q6BXI8, G2R9I6 and U9TUL6. Evenmore preferably, the mutant polypeptide having malate dehydrogenaseactivity is a mutant S. cerevisiae peroxisomal NAD-MDH (MDH3).

Additionally, in the recombinant host cell of the invention, the mutantpolypeptide having malate dehydrogenase activity may be a mutant of ahomologous or heterologous NAD(P)-malate dehydrogenase. In a preferredembodiment, the MDH mutant is a mutant of a homologous NAD(P)-malatedehydrogenase. More preferably, the MDH mutant is a mutant of ahomologous NAD(H)-malate dehydrogenase (EC 1.1.1.37). More preferably,the mutant polypeptide having malate dehydrogenase activity is a mutantof a homologous peroxisomal NAD(H)-malate dehydrogenase.

In this context, the term “homologous” or “endogenous” when used toindicate the relation between a given (recombinant) nucleic acid orpolypeptide molecule and a given host organism or host cell, isunderstood to mean that in nature the nucleic acid or polypeptidemolecule is produced by a host cell or organism of the same species,preferably of the same variety or strain.

The term “heterologous” as used herein refers to nucleic acid or aminoacid sequences not naturally occurring in a host cell. In other words,the nucleic acid or amino acid sequence is not identical to thatnaturally found in the host cell.

Preferably, in a recombinant host cell of the present invention, thenucleic sequence encoding said mutant polypeptide having malatedehydrogenase activity is expressed in the cytosol and the mutantpolypeptide having malate dehydrogenase activity is active in thecytosol. In some instances, cytosolic expression may be obtained bydeletion of a peroxisomal or mitochondrial targeting signal. Thepresence of a peroxisomal or mitochondrial targeting signal may forinstance be determined by the method disclosed by Schluter et al.,Nucleid Acid Research 2007, 35, D815-D822. When the MDH mutant is amutant S. cerevisiae peroxisomal NAD-MDH (e.g. a mutant MDH3), itsC-terminal SKL is preferably deleted such that it is active in thecytosol.

Typically, the mutant polypeptide having malate dehydrogenase activitymay have at least about 40%, 50%, 60%, 70%, 80% sequence identity with areference MDH polypeptide, such as the MDH of SEQ ID NO: 53 or SEQ IDNO: 39, for example at least 85% sequence identity with a reference MDHpolypeptide, such as at least about 90% sequence identity with areference MDH polypeptide, at least 95% sequence identity with areference MDH polypeptide, at least 98% sequence identity with areference MDH polypeptide or at least 99% sequence identity with areference MDH polypeptide.

It has been surprisingly found that said mutant MDH polypeptide asdescribed herein above confers an increase in the production of adicarboxylic acid in a recombinant host cell when said mutant is(over)expressed in said recombinant host cell as compared to theproduction level of an equivalent recombinant host cell which(over)expresses a reference polypeptide having MDH activity; the“reference MDH polypeptide” being typically a NAD-MDH (EC 1.1.1.37), forexample a malate dehydrogenase having an amino acid sequence set out inSEQ ID NO: 39.

Accordingly, there is thus provided a recombinant host cell which iscapable of producing or produces a dicarboxylic acid and which comprisesa nucleic acid sequence encoding a mutant polypeptide having malatedehydrogenase activity as described herein above.

A recombinant host cell of the invention is capable of producing orproduces a dicarboxylic acid, such as malic acid, fumaric acid and/orsuccinic acid.

The terms “dicarboxylic acid” and “dicarboxylate”, such as “succinicacid” and “succinate”, have the same meaning herein and are usedinterchangeably, the first being the hydrogenated form of the latter.

Typically, the recombinant host cell of the invention will produce anincreased amount of a dicarboxylic acid in comparison to a recombinanthost cell expressing a reference MDH polypeptide, for example that ofSEQ ID NO: 39. The production of a dicarboxylic acid may be increased,by at least 5%, 10%, at least 20%, at least 30%, at least 40% at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 100% or more. Production level may be expressed interms of g/L, so an increase in the production level of a dicarboxylicacid will be evident by higher level of production in terms of g/L.

The recombinant host cell of the invention or a parent of said host cellmay be any type of host cell. Accordingly, both prokaryotic andeukaryotic cells are included. Host cells may also include, but are notlimited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS,MDCK, 293, 3T3, WI38, and choroid plexus cell lines.

A suitable host cell of the invention may be a prokaryotic cell.Preferably, the prokaryotic cell is a bacterial cell. The term“bacterial cell” includes both Gram-negative and Gram-positivemicroorganisms.

Suitable bacteria may be selected from e.g. Escherichia, Actinobacillus,Anabaena, Caulobactert, Gluconobacter, Mannheimia, Basfia, Rhodobacter,Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium,Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter,Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus orActinomycetes such as Streptomyces and Actinoplanes species. Preferably,the bacterial cell is selected from the group consisting of Bacillussubtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B.megaterium, B. halodurans, B. pumilus, Actinobacillus succinogenes,Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacteriumextorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens,Pseudomonas putida, Pseudomonas fluorescens, Paracoccus denitrificans,Escherichia coli, Corynebacterium glutamicum, Mannheimiasuccinoproducens, Basfia succinoproducens, Staphylococcus carnosus,Streptomyces lividans, Streptomyces clavuligerus, Sinorhizobium meliotiand Rhizobium radiobacter.

A host cell according to the invention may be a eukaryotic host cell.Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal,or algal cell. More preferably, the eukaryotic cell is a fungal cell. Asuitable fungal cell may for instance belong to genera Saccharomyces,Schizosaccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces,Yarrowia, Candida, Hansenula, Humicola, Pichia, Issatchenkia, Kloeckera,Schwanniomyces, Torulaspora, Trichosporon, Brettanomyces, Rhizopus,Zygosaccharomyces, Pachysolen or Yamadazyma. A fungal cell may forinstance belong to a species of Saccharomyces cerevisiae, S. uvarum, S.bayanus S. pastorianus, S. carlsbergensis, Aspergillus niger,Penicillium chrysogenum, Pichia stipidis, P. pastoris, Kluyveromycesmarxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candidasonorensis, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis, C.kruisei, C. glabrata, Hansenula polymorpha, Issatchenkia orientalis,Torulaspora delbrueckii, Brettanomyces bruxellensis, Rhizopus oryzae orZygosaccharomyces bailii. In one embodiment, a fungal cell of thepresent invention is a yeast, for instance belonging to a Saccharomycessp., such as a Saccharomyces cerevisiae.

Examples of specific host yeast cells include C. sonorensis, K.marxianus, K. thermotolerans, C. methanesorbosa, Saccharomyces bulderi(S. bulden), P. kudriavzevii, I. orientalis, C. lambica, C. sorboxylosa,C. zemplinina, C. geochares, P. membranifaciens, Z. kombuchaensis, C.sorbosivorans, C. vanderwaltii, C. sorbophila, Z. bisporus, Z. lentus,Saccharomyces bayanus (S. bayanus), D. castellii, C, boidinii, C.etchellsii, K. lactis, P. jadinii, P. anomala, Saccharomyces cerevisiae(S. cerevisiae), Pichia galeiformis, Pichia sp. YB-4149 (NRRLdesignation), Candida ethanolica, P. deserticola, P. membranifaciens, P.fermentans and Saccharomycopsis crataegensis (S. crataegensis). Suitablestrains of K. marxianus and C. sonorensis include those described in WO00/71738 A1, WO 02/42471 A2, WO 03/049525 A2, WO 03/102152 A2 and WO03/102201A2. Suitable strains of I. orientalis are ATCC strain 32196 andATCC strain PTA-6648. In the invention, the host cell may be a Crabtreenegative as a wild-type strain. The Crabtree effect is defined as theoccurrence of fermentative metabolism under aerobic conditions due tothe inhibition of oxygen consumption by a microorganism when cultured athigh specific growth rates (long-term effect) or in the presence of highconcentrations of glucose (short-term effect). Crabtree negativephenotypes do not exhibit this effect, and are thus able to consumeoxygen even in the presence of high concentrations of glucose or at highgrowth rates.

The eukaryotic cell may be a filamentous fungal cell. Filamentous fungiinclude all filamentous forms of the subdivision Eumycota and Oomycota(as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionaryof The Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK). The filamentous fungi are characterized by a mycelialwall composed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. Filamentous fungal strainsinclude, but are not limited to, strains of Acremonium, Aspergillus,Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium,Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor,Myceliophthora, Mortierella, Neocaffimastix, Neurospora, Paecilomyces,Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus,Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus,Thielavia, Tolypocladium, Trametes and Trichoderma. Preferredfilamentous fungal strains that may serve as host cells belong to thespecies Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus,Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum,Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromycesemersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtorathermophyla. Reference host cells for the comparison of fermentationcharacteristics of transformed and untransformed cells, include e.g.Aspergillus niger CBS120.49, CBS 513.88, Aspergillus oryzae ATCC16868,ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601,ATCC12892, Aspergillus fumigatus AF293 (CBS101355), P. chrysogenum CBS455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2,Thielavia terrestris NRRL8126, Talaromyces emersonii CBS 124.902,Rasamsonia emersonii CBS393.64, Acremonium chrysogenum ATCC 36225, ATCC48272, Trichoderma reesei ATCC 26921, ATCC 56765, ATCC 26921,Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006 andderivatives of all of these strains.

A more preferred host cell belongs to the genus Aspergillus, morepreferably the host cell belongs to the species Aspergillus niger. Whenthe host cell according to the invention is an Aspergillus niger hostcell, the host cell preferably is CBS 513.88, CBS124.903 or a derivativethereof.

In a preferred embodiment, a host cell according to the invention is ayeast cell selected from the group consisting of Candida, Hansenula,Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,or Yarrowia strains, or a filamentous fungal cell selected from thegroup consisting of filamentous fungal cells belong to a species ofAcremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium,Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma.

A host cell of the invention may be any wild type strain producing adicarboxylic acid. Furthermore, a suitable host cell may be a cell whichhas been obtained and/or improved by subjecting a parental or wild typecell of interest to a classical mutagenic treatment or to recombinantnucleic acid transformation. Thus, a suitable host cell may already becapable of producing the dicarboxylic acid. However, the cell may alsobe provided with a homologous or heterologous expression construct thatencodes one or more polypeptides involved in the production of thedicarboxylic acid.

Accordingly, in some embodiments, a recombinant host cell of theinvention may comprise a MDH mutant polypeptide and an active reductivetricarboxylic acid (TCA) pathway from phosphoenolpyruvate or pyruvate tosuccinate.

Accordingly, in addition to a nucleic acid encoding a MDH mutantpolypeptide, a host cell of the invention may comprise a nucleotidesequence comprising sequence encoding one or more of a pyruvatecarboxylase, a phosphoenolpyruvate carboxykinase, a phosphoenolpyruvatecarboxylase, a malate dehydrogenase, a fumarase, an isocitrate lyase, amalate synthase, a fumarate reductase and/or a dicarboxylic acidtransporter. Preferably, one or more such enzymes are (over)expressedand active in the cytosol.

Thus, a recombinant host cell of the invention may overexpress asuitable homologous or heterologous nucleotide sequence that encodes aendogenous and/or heterologous enzyme that catalyzes a reaction in thecell resulting in an increased flux towards a dicarboxylic acid suchmalic acid, fumaric acid and/or succinic acid.

A recombinant host cell of the invention may overexpress an endogenousor heterologous nucleic acid sequence as described herein below.

A recombinant host cell of the invention may comprise a geneticmodification with a pyruvate carboxylase (PYC), that catalyses thereaction from pyruvate to oxaloacetate (EC 6.4.1.1). The pyruvatecarboxylase may for instance be active in the cytosol upon expression ofthe gene. For instance, the host cell overexpresses a pyruvatecarboxylase, for instance an endogenous or homologous pyruvatecarboxylase is overexpressed. The recombinant fungal host cell accordingto the present invention may be genetically modified with a pyruvatecarboxylase which has at least 70%, preferably at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity with amino acid sequence encoded by the nucleic acid sequenceof SEQ ID NO: 46.

Preferably, the recombinant host cell expresses a nucleotide sequenceencoding a phosphoenolpyruvate (PEP) carboxykinase in the cytosol.Preferably a nucleotide sequence encoding a PEP carboxykinase isoverexpressed. The PEP carboxykinase (EC 4.1.1.49) preferably is aheterologous enzyme, preferably derived from bacteria, more preferablythe enzyme having PEP carboxykinase activity is derived from Escherichiacoli, Mannheimia sp., Actinobacillus sp., or Anaerobiospirillum sp.,more preferably Mannheimia succiniciproducens. A gene encoding a PEPcarboxykinase may be overexpressed and active in the cytosol of a fungalcell. Preferably, a recombinant fungal cell according to the presentinvention is genetically modified with a PEP carboxykinase which has atleast 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acidsequence of SEQ ID NO: 47.

Preferably, the recombinant host cell expresses a nucleotide sequenceencoding a phosphoenolpyruvate (PEP) carboxylase in the cytosol.Preferably a nucleotide sequence encoding a PEP carboxylase isoverexpressed. The PEP carboxylase (EC 4.1.1.31) preferably is aheterologous enzyme, preferably derived from bacteria.

In one embodiment, the recombinant host cell is further geneticallymodified with a gene encoding a malate dehydrogenase (MDH) active in thecytosol upon expression of the gene. Cytosolic expression may beobtained by deletion of a peroxisomal targeting signal. The malatedehydrogenase may be overexpressed. A cytosolic MDH may be any suitablehomologous or heterologous malate dehydrogenase, catalyzing the reactionfrom oxaloacetate to malate (EC 1.1.1.37), for instance derived from S.cerevisiae.

Preferably, the MDH is S. cerevisiae MDH3, more preferably one which hasa C-terminal SKL deletion such that it is active in the cytosol.Preferably, the recombinant fungal cell according to the presentinvention comprises a nucleotide sequence encoding a malatedehydrogenase that has at least 70%, preferably at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity with the amino acid sequence of SEQ ID NO: 39.

In another embodiment, the recombinant host cell of the presentdisclosure is further genetically modified with a gene encoding afumarase, that catalyses the reaction from malic acid to fumaric acid(EC 4.2.1.2). A gene encoding fumarase may be derived from any suitableorigin, preferably from microbial origin, for instance a yeast such asSaccharomyces or a filamentous fungus, such Rhizopus oryzae, or abacterium such a Escherichia coli. The host cell of the presentdisclosure may overexpress a nucleotide sequence encoding a fumarase.The fumarase may be active in the cytosol upon expression of thenucleotide sequence, for instance by deleting a peroxisomal targetingsignal. It was found that cytosolic activity of a fumarase resulted in ahigh productivity of a dicarboxylic acid by a fungal cell.

Preferably, the recombinant host cell of the present inventionoverexpresses a nucleotide sequence encoding a fumarase that has atleast 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acidsequence of SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 67.

In another embodiment, the recombinant host cell is genetically modifiedwith any suitable heterologous or homologous gene encoding aNAD(H)-dependent fumarate reductase, catalyzing the reaction fromfumarate to succinate (EC 1.3.1.6). The NAD(H)-dependent fumaratereductase may be a heterologous enzyme, which may be derived from anysuitable origin, for instance bacteria, fungi, protozoa or plants. Afungal cell of the present disclosure comprises a heterologousNAD(H)-dependent fumarate reductase, preferably derived from aTrypanosoma sp, for instance a Trypanosoma brucei. In one embodiment,the NAD(H)-dependent fumarate reductase is expressed and active in thecytosol, for instance by deleting a peroxisomal targeting signal. Thehost cell may overexpress a gene encoding a NAD(H)-dependent fumaratereductase.

Preferably, the recombinant host cell according to the present inventionis genetically modified with a NAD(H)-dependent fumarate reductase,which has at least at least 70%, preferably at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identitywith the amino acid sequence of SEQ ID NO: 8. Also preferably, the hostcell according to the present invention is genetically modified with avariant polypeptide having fumarate reductase activity as disclosed inWO2015/086839.

In another embodiment, the recombinant host cell of the inventionexpresses a nucleotide sequence encoding a dicarboxylic acid transporterprotein. Preferably the dicarboxylic acid transporter protein isoverexpressed. A dicarboxylic acid transporter protein may be anysuitable homologous or heterologous protein. Preferably the dicarboxylicacid transporter protein is a heterologous protein. A dicarboxylic acidtransporter protein may be derived from any suitable organism,preferably from yeast or fungi such as Schizosaccharomyces pombe orAspergillus niger. Preferably, a dicarboxylic acid transporter proteinis a dicarboxylic acid transporter/malic acid transporter protein, eg.from Aspergillus niger which at least 70%, preferably at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity with the amino acid sequence of SEQ ID NO: 50.

The recombinant host cell may further comprise a genetic modificationwith a gene encoding an isocitrate lyase (EC 4.1.3.1), which may be anysuitable heterologous or homologous enzyme. The isocitrate lyase may forinstance be obtained from Kluyveromyces lactis or Escherichia coli.

The recombinant host according to the present invention is geneticallymodified with a isocitrate lyase which has at least 70%, preferably atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity with amino acid sequence encoded by the nucleicacid sequence of SEQ ID NO: 51.

The recombinant host cell may further comprise a genetic modificationwith a malate synthase (EC 2.3.3.9). The malate synthase may beoverexpressed and/or active in the cytosol, for instance by deletion ofa peroxisomal targeting signal. In the event the malate synthase is a S.cerevisiae malate synthase, for instance the native malate synthase isaltered by the deletion of the SKL carboxy-terminal sequence.

The recombinant host cell of the present invention is geneticallymodified with a malate synthase which at least 70%, preferably at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity with amino acid sequence encoded by the nucleic acidsequence of SEQ ID NO: 52.

In another embodiment, the recombinant host cell of the inventiondisclosed herein comprises a disruption of a gene a pyruvatedecarboxylase (EC 4.1.1.1), catalyzing the reaction from pyruvate toacetaldehyde.

In another embodiment, the recombinant host cell of the invention maycomprise a disruption of a gene encoding an enzyme of the ethanolfermentation pathway. A gene encoding an enzyme of an ethanolfermentation pathway, may be pyruvate decarboxylase (EC 4.1.1.1),catalyzing the reaction from pyruvate to acetaldehyde, or alcoholdehydrogenase (EC 1.1.1.1), catalyzing the reaction from acetaldehyde toethanol. Preferably, a host cell of the invention comprises a disruptionof one, two or more genes encoding an alcohol dehydrogenase. In theevent the fungal cell is a yeast, e.g. S. cerevisiae, the yeastpreferably comprises a disruption of one or more alcohol dehydrogenasegenes (adh1 adh2, adh3, adh4, adh5, adh6).

Alternatively or in addition, the recombinant host cell of the inventionmay comprise at least one gene encoding glycerol-3-phosphatedehydrogenase which is not functional. A glycerol-3-phosphatedehydrogenase gene that is not functional is used herein to describe aeukaryotic cell, which comprises a reduced glycerol-3-phosphatedehydrogenase activity, for instance by mutation, disruption, ordeletion of the gene encoding glycerol-3-phosphate dehydrogenase,resulting in a decreased formation of glycerol as compared to awild-type cell. In the event the fungal cell is a yeast, e.g. S.cerevisiae, the yeast preferably comprises a disruption of one or moreglycerol-3-phosphate dehydrogenase genes (gpd1, gpd2, gut2).

Alternatively or in addition to the above, the recombinant host cell ofthe invention may comprise at least one gene encoding a mitochondrialexternal NADH dehydrogenase which is not functional. A mitochondrialexternal NADH dehydrogenase gene that is not functional is used hereinto describe a eukaryotic cell, which comprises a reduced NADHdehydrogenase activity, for instance by mutation, disruption, ordeletion of the gene encoding the mitochondrial external NADHdehydrogenase. In the event the fungal cell is a yeast, e.g. S.cerevisiae, the yeast preferably comprises a disruption of one or moremitochondrial external NADH dehydrogenase genes (nde1, nde2).

Alternatively or in addition to the above, the recombinant host cell ofthe invention may comprise at least one gene encoding an aldehydedehydrogenase which is not functional. An aldehyde dehydrogenase genethat is not functional is used herein to describe a eukaryotic cell,which comprises a reduced aldehyde dehydrogenase activity, for instanceby mutation, disruption, or deletion of the gene encoding the aldehydedehydrogenase. In the event the fungal cell is a yeast, e.g. S.cerevisiae, the yeast preferably comprises a disruption of one or morealdehyde dehydrogenase genes (ald2, ald3, ald4, ald5, ald6).

Preferably, the recombinant host cell of the present invention is arecombinant fungal cell. More preferably, the host cell of the presentinvention is a recombinant yeast cell. Preferred embodiments of therecombinant fungal cell or recombinant yeast cell are as describedherein above for the recombinant host cell.

In some embodiments of the invention, the recombinant host cell is arecombinant yeast cell which is capable of producing a dicarboxylic acidas described herein above and which comprises a nucleic acid sequenceencoding a mutant polypeptide having malate dehydrogenase activity asdetailed herein above. Said MDH mutant may be a mutant of a homologousor heterologous wild-type MDH polypeptide. In a preferred embodiment,said MDH mutant is a mutant of a homologous MDH polypeptide. In an evenmore preferred embodiment, the recombinant yeast cell is a recombinantSaccharomyces, for example S. cerevisiae, and the MDH mutant is a mutantof a homologous MDH, for example MDH2 or MDH3. In a more specificembodiment, said recombinant yeast cell comprises a nucleic sequenceencoding a mutant polypeptide having malate dehydrogenase activity asdefined in Table 1 and wherein the amino acid residue corresponding toamino acid 34 in SEQ ID NO: 39 is selected from glycine (G) or serine(S).

Standard genetic techniques, such as overexpression of enzymes in thehost cells, genetic modification of host cells, or hybridisationtechniques, are known methods in the art, such as described in Sambrooket al. (Molecular Cloning: A Laboratory Manual, 3^(rd) edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y,2001) or Ausubel et al. (Current protocols in molecular biology, GreenPublishing and Wiley Interscience, N Y, 1987). Methods fortransformation, genetic modification of fungal host cells are known frome.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671,WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No. 6,265,186.

As used herein, the terms “nucleic acid”, “polynucleotide” or “nucleicacid molecule” are intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA. The nucleic acid may be synthesized using oligonucleotide analogsor derivatives (e.g., inosine or phosphorothioate nucleotides). Sucholigonucleotides can be used, for example, to prepare nucleic acids thathave altered base-pairing abilities or increased resistance tonucleases.

The term “nucleic acid construct” is herein referred to as a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally-occurring gene or, more typically, which has been modifiedto contain segments of nucleic acid which are combined and juxtaposed ina manner which would not otherwise exist in nature. The term nucleicacid construct is synonymous with the term “expression cassette” whenthe nucleic acid construct contains all the control sequences requiredfor expression of a coding sequence in a host cell, wherein said controlsequences are operably linked to said coding sequence.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements (or coding sequences or nucleic acid sequence)in a functional relationship. A nucleic acid sequence is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For instance, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thecoding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequencesknown to one of skilled in the art. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is active underenvironmental or developmental regulation.

A promoter that could be used to achieve the expression of a nucleotidesequence coding for an enzyme such a malate dehydrogenase or any otherenzyme introduced in the host cell of the invention, may be not nativeto a nucleotide sequence coding for the enzyme to be expressed, i.e. apromoter that is heterologous to the nucleotide sequence (codingsequence) to which it is operably linked. Preferably, the promoter ishomologous, i.e. endogenous to the host cell.

Suitable promoters in this context include both constitutive andinducible natural promoters as well as engineered promoters, which arewell known to the person skilled in the art. Suitable promoters ineukaryotic host cells may be GAL7, GAL10, or GAL 1, CYC1, HIS3, ADH1,PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and AOX1. Othersuitable promoters include PDC, GPD1, PGK1, TEF1, and TDH.

Usually a nucleotide sequence encoding an enzyme comprises a“terminator”. Any terminator, which is functional in the eukaryoticcell, may be used in the present invention. Preferred terminators areobtained from natural genes of the host cell. Suitable terminatorsequences are well known in the art. Preferably, such terminators arecombined with mutations that prevent nonsense mediated mRNA decay in thehost cell of the invention (see for example: Shirley et al., 2002,Genetics 161:1465-1482).

The nucleic acid construct may be incorporated into a “vector”, such asan expression vector and/or into a host cell in order to effectexpression of the polypeptide to be expressed.

The expression vector may be any vector (e.g., a plasmid or virus),which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the polynucleotide encoding thepolypeptide having malate dehydrogenase activity. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i. e., a vector, which exists as anextra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extra-chromosomal element,a mini-chromosome, or an artificial chromosome. If intended for use in ahost cell of fungal origin, a suitable episomal nucleic acid constructmay e.g. be based on the yeast 2p or pKD1 plasmids (Gleer et al., 1991,Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995,Curr Genet. 29:482-489).

Alternatively, the expression vector may be one which, when introducedinto the host cell, is integrated into the genome and replicatedtogether with the chromosome(s) into which it has been integrated. Theintegrative cloning vector may integrate at random or at a predeterminedtarget locus in the chromosomes of the host cell. In a preferredembodiment of the invention, the integrative cloning vector comprises aDNA fragment, which is homologous to a DNA sequence in a predeterminedtarget locus in the genome of host cell for targeting the integration ofthe cloning vector to this predetermined locus. In order to promotetargeted integration, the cloning vector is preferably linearized priorto transformation of the cell. Linearization is preferably performedsuch that at least one but preferably either end of the cloning vectoris flanked by sequences homologous to the target locus. The length ofthe homologous sequences flanking the target locus is preferably atleast 20 bp, at least 30 bp, at least 50 bp, at least 0.1 kb, at least0.2 kb, at least 0.5 kb, at least 1 kb, at least 2 kb or longer. Theefficiency of targeted integration into the genome of the host cell,i.e. integration in a predetermined target locus, is increased byaugmented homologous recombination abilities of the host cell.

The homologous flanking DNA sequences in the cloning vector, which arehomologous to the target locus, are derived from a highly expressedlocus meaning that they are derived from a gene, which is capable ofhigh expression level in the host cell. A gene capable of highexpression level, i.e. a highly expressed gene, is herein defined as agene whose mRNA can make up at least 0.5% (w/w) of the total cellularmRNA, e.g. under induced conditions, or alternatively, a gene whose geneproduct can make up at least 1% (w/w) of the total cellular protein, or,in case of a secreted gene product, can be secreted to a level of atleast 0.1 g/l.

A nucleic acid construct or expression vector may be assembled in vivoin a host cell of the invention and, optionally, integrated into thegenome of the cell in a single step (see, for example, WO2013/076280)

More than one copy of a nucleic acid construct or expression vector ofthe invention may be inserted into the host cell to increase productionof the polypeptide having malate dehydrogenase activity(over-expression) encoded by the nucleic acid sequence comprised withinthe nucleic acid construct. This can be done, preferably by integratinginto its genome two or more copies of the nucleic acid, more preferablyby targeting the integration of the nucleic acid at a highly expressedlocus defined as defined above.

It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc.

A nucleic acid construct and/or expression vector of the invention canbe introduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell well known to those skilled in the art. Suitablemethods for transforming or transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 3rd edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y,2001), Davis et al. (Basic Methods in Molecular Biology, 1^(st) edition,Elsevier, 1986) and other laboratory manuals.

Cytosolic expression of the enzymes described above may be obtained bydeletion of a peroxisomal or mitochondrial targeting signal. Thepresence of a peroxisomal or mitochondrial targeting signal may forinstance be determined by the method disclosed by Schlüter et al.(Schluter et al., 2007, Nucleic Acid Research 35: D815-D822).

A comparison of sequences and determination of percentage of sequenceidentity between two sequences can be accomplished using a mathematicalalgorithm. The skilled person will be aware of the fact that severaldifferent computer programs are available to align two sequences anddetermine the identity between two sequences (Kruskal, J. B. (1983) Anoverview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.),Time warps, string edits and macromolecules: the theory and practice ofsequence comparison, pp. 1-44 Addison Wesley). The percent sequenceidentity between two amino acid sequences or between two nucleotidesequences may be determined using the Needleman and Wunsch algorithm forthe alignment of two sequences. (Needleman, S. B. and Wunsch, C. D.(1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences andnucleotide sequences can be aligned by the algorithm. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this invention the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 isused for the substitution matrix. For nucleotide sequence, EDNAFULL isused. The optional parameters used are a gap-open penalty of 10 and agap extension penalty of 0.5. The skilled person will appreciate thatall these different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentageof sequence identity between a query sequence and a sequence of theinvention is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid or identical nucleotide inboth sequences divided by the total length of the alignment aftersubtraction of the total number of gaps in the alignment. The identitydefined as herein can be obtained from NEEDLE by using the NOBRIEFoption and is labeled in the output of the program as“longest-identity”.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the blastn and blastxprograms (version 2.2.31 or above) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theblastn program, score=100, word-size=11 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the blastx program, score=50, word-size=3to obtain amino acid sequences homologous to protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,blastx and blastn) can be used. See the homepage of the National Centerfor Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

According to the present invention, there is also provided a process forthe production of a dicarboxylic acid, such as succinic acid, whichprocess comprises fermenting the recombinant host cell of the inventionas described herein above, under conditions suitable for production ofthe dicarboxylic acid, and optionally, recovering the dicarboxylic acidfrom the fermentation medium.

In the process, the recombinant host cell of the invention is fermentedin a vessel comprising a suitable fermentation medium. The termfermenting, fermentation or fermented and the like as used herein refersto the microbial production of compounds, here dicarboxylic acids fromcarbohydrates.

Preferably, the fermentation product is a dicarboxylic acid, preferablymalic acid, fumaric acid and/or succinic acid, preferably succinic acid.

A batch fermentation is defined herein as a fermentation wherein allnutrients are added at the start of a fermentation.

A fed-batch fermentation is a batch fermentation wherein the nutrientsare added during the fermentation. Products in a batch and fed-batchfermentation may be harvested at a suitable moment, for instance whenone or more nutrients are exhausted.

A continuous fermentation is a fermentation wherein nutrients arecontinuously added to the fermentation and wherein products arecontinuously removed from the fermentation.

In one embodiment fermenting the host cell in the process of theinvention is carried out under carbohydrate limiting conditions. As usedherein, carbohydrate limiting conditions are defined as maintaining thecarbohydrate concentration below 10 g/l, for example about 5 g/l.

The process for the production of dicarboxylic acid according to thepresent invention may be carried out in any suitable volume and scale,preferably on an industrial scale. Industrial scale is defined herein asa volume of at least 10, or 100 litres, preferably at least 1 cubicmetre, preferably at least 10, or 100 cubic metres, preferably at least1000 cubic metres, usually below 10,000 cubic metres.

Fermenting the recombinant host cell in the process of the invention maybe carried out in any suitable fermentation medium comprising a suitablenitrogen source, carbohydrate and other nutrients required for growthand production of a dicarboxylic acid in the process of the invention. Asuitable carbohydrate in the fermentation process according to theinvention may be glucose, galactose, xylose, arabinose, sucrose, ormaltose.

In one embodiment, the fermentation process is carried out under apartial CO₂ pressure of between 5% and 60%, preferably about 50%.

The pH during the process for the production of dicarboxylic acidusually lowers during the production of the dicarboxylic acid.Preferably, the pH in the process for the production of dicarboxylicacid ranges between 1 and 5, preferably between 1.5 and 4.5, morepreferably between 2 and 4.

In another preferred embodiment, the process according to the presentinvention comprises a step of preculturing the host cell under aerobicconditions in the presence of a carbohydrate. Preferably, thefermentation of the host cell during preculturing is carried out at a pHof between 4 and 6. Preferably, the carbohydrate during preculturing isa non-repressing carbohydrate, preferably galactose. It has been foundadvantageous to preculture host cells on a non-repressing carbohydrate,since this prevents glucose repression occurring, which may negativelyinfluence the amount of biomass produced. In addition, it has been foundthat a step of preculturing host cells under aerobic conditions resultsin a higher biomass yield and a faster growth. Preferably, thepreculturing is carried out in batch mode.

A propagation step for producing increased biomass is typically carriedout, preferably under carbohydrate limiting conditions.

The process for producing a dicarboxylic acid may be carried out at anysuitable temperature. A suitable temperature may for instance be betweenabout 10 and about 40 degrees Celsius, for instance between about 15 andabout 30 degrees Celsius.

In an embodiment, the process of the invention is carried out in such away that at least a portion of the host cells is reused, i.e. recycled.The cells may be recycled back into the original vessel or into a secondvessel. Preferably, the medium into which the recycled host cells areintroduced is supplemented with a vitamin and/or a trace element.

In a preferred embodiment, the fermentation medium comprises an amountof succinic acid of between 1 and 150 g/l, preferably between 5 and 100g/l, more preferably between 10 and 80 g/l or between 15 and 60 g/l ofsuccinic acid. In any event, the recombinant host cell of the inventionwill typically be capable of accumulating more succinic acid in thefermentation medium as compared to a host cell that has been modifiedwith a reference MDH polypeptide, for example that of SEQ ID NO: 39.

The process for the production of a dicarboxylic acid may furthercomprise recovering the dicarboxylic acid. Recovery of the dicarboxylicacid may be carried out by any suitable method known in the art, forinstance by crystallization, ammonium precipitation, ion exchangetechnology, centrifugation or filtration or any suitable combination ofthese methods.

In a preferred embodiment, the recovery of the dicarboxylic acidcomprises crystallizing the dicarboxylic acid and forming dicarboxylicacid crystals. Preferably, the crystallizing of the dicarboxylic acidcomprises removing part of the fermentation medium, preferably byevaporation, to obtain a concentrated medium.

According to the present invention, the dicarboxylic acid, such assuccinic acid may be recovered by crystallizing the dicarboxylic acid,such as succinic acid, from an aqueous solution having a pH of between 1and 5 and comprising succinic acid, comprising evaporating part of theaqueous solution to obtain a concentrated solution, lowering thetemperature of the concentrated solution to a value of between 5 and 35degrees Celsius, wherein succinic acid crystals are formed. Preferably,the crystallizing comprises bringing the temperature of the concentratedmedium to a temperature of between 10 and 30 degrees Celsius, preferablybetween 15 and 25 degrees Celsius. Preferably, the fermentation mediumhas a pH of between 1.5 and 4.5, preferably between 2 and 4.

It has been found that crystallizing the dicarboxylic acid, such assuccinic acid, at higher temperatures such as between 10 and 30 degreesCelsius results in crystals of a dicarboxylic acid, such as succinicacid, with a lower amount of impurities such as organic acid, protein,color and/or odor, than crystals of a dicarboxylic acid, such assuccinic acid, that were crystallized at a low temperature of below 10degrees.

Another advantage of crystallizing succinic acid at a higher temperatureis that it requires a lower amount of energy for cooling the aqueoussolution as compared to a process wherein crystallizing the dicarboxylicacid is carried out below 10 or 5 degrees Celsius, resulting in a moreeconomical and sustainable process.

Preferably, the crystallizing of the dicarboxylic acid, such as succinicacid, comprises a step of washing the dicarboxylic acid crystals.Dicarboxylic acid, such as succinic acid, may be crystallized directlyfrom the fermentation medium having a pH of between 1 and 5 to a purityof at least 90% w/w, preferably at least 95, 96, 97, or at least 98%, or99 to 100% w/w.

In a preferred embodiment, the process for the production of adicarboxylic acid further comprises using the dicarboxylic acid in anindustrial process.

Preferably, the dicarboxylic acid, such as succinic acid, that isprepared in the process according to the present invention is furtherconverted into a desirable product. A desirable product may for instancebe a polymer, such as polybutylene succinic acid (PBS), a deicing agent,a food additive, a cosmetic additive or a surfactant. That is to say,the invention provides a method for the production of a product, forexample, a polymer, such as polybutylene succinic acid (PBS), a deicingagent, a food additive, a cosmetic additive or a surfactant, whichmethod comprises: producing a dicarboxylic acid as described herein; andusing said dicarboxylic acid in the production of said product.

A reference herein to a patent document or other matter which is givenas prior art is not to be taken as an admission that that document ormatter was known or that the information it contains was part of thecommon general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES General Materials and Methods

DNA Procedures

Standard DNA procedures were carried out as described elsewhere(Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2^(nd)Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)unless otherwise stated. DNA was amplified using the proofreading enzymePhusion polymerase (New England Biolabs, USA) according tomanufacturer's instructions. Restriction enzymes were from Invitrogen orNew England Biolabs.

Microtiter Plate (MTP) Fermentation of Dicarboxylic Acid ProductionStrains

To determine dicarboxylic acid production, strains were grown intriplicate in micro titer plates in humidity shakers (Infors) for 3 daysat 30 degrees at 550 rpm and 80% humidity. The medium was based onVerduyn medium (Verduyn C, Postma E, Scheffers W A, Van Dijken J P.Yeast, 1992 July; 8(7):501-517), but modifications in carbon andnitrogen source were made as described herein below.

MTP pre-culture medium composition Concentration Raw material (g/l)Galactose C₆H₁₂O₆•H₂O 40.0 Urea (NH₂)₂CO 2.3 Potassium KH₂PO₄ 3.0dihydrogen phosphate Magnesium sulphate MgSO₄•7H₂O 0.5 Trace element 1solution^(a) Vitamin solution^(b) 1 Component Formula Concentration(g/kg) EDTA C₁₀H₁₄N₂Na₂O₈•2H₂O 15.00 Zincsulphate•7H₂O ZnSO₄•7H₂O 4.50Manganese- MnCl₂•2H₂O 0.84 chloride•2H₂O Cobalt (II) CoCl₂•6H₂O 0.30chloride•6H₂O Copper (II) CuSO₄•5H₂O 0.30 sulphate•5H₂O SodiumNa₂MoO₄•2H₂O 0.40 molybdenum•2H₂O Calcium- CaCl₂•2H₂O 4.50 chloride•2H₂OIronsulphate•7H₂O FeSO₄•7H₂O 3.00 Boric acid H₃BO₃ 1.00 Potassium iodideKl 0.10 Biotin (D−) C₁₀H₁₆N₂O₃S 0.05 Ca D(+) C₁₈H₃₂CaN₂O₁₀ 1.00panthothenate Nicotinic acid C₆H₅NO₂ 1.00 Myo-inositol C₆H₁₂O₆ 25.00Thiamine C₁₂H₁₈Cl₁₂N₄OS•xH₂O 1.00 chloride hydrochloride PyridoxolC₈H₁₂CINO₃ 1.00 hydrochloride p-aminobenzoic acid C₇H₇NO₂ 0.20 ^(a)Traceelements solution ^(b)Vitamin solution

80 microliters of pre-culture was used to inoculate 2.5 ml of mediumwith 1.5% galactose as carbon source in 24-well plates. The cultureswere grown for 72 hours in humidity shakers (Infors) at 30° C., 550 rpm,80% humidity. After generating biomass, a production experiment wasstarted by re-suspending cells into 2.5 ml of mineral medium withglucose as carbon source. The main cultures were incubated in humidityshakers (Infors) at 30 degrees at 550 rpm and 80% humidity and sampleswere taken after 48 hours of cultivation.

Metabolite Analysis of MTP Samples by NMR

For metabolite analysis of MTP samples, 90 microliter of supernatant offermentation samples is mixed with 10 microliter of NMR standard (20 g/lmaleic acid) and 100 microliter of 10% D₂O solution. The samples arelyophilized and subsequently dissolved in 1 mL D₂O.

1D 1H NMR spectra are recorded on a BEST Bruker Avance III spectrometer,operating at a proton frequency of 500 MHz, equipped with a He-cooledcryo probe, using a pulse program without water suppression (ZG) at atemperature of 300 K, with a 90 degree excitation pulse, acquisitiontime of 2.0 seconds and a relaxation delay of 40 seconds. The number ofscans was set at 8.

The malic acid concentration [in g/l] is calculated based on thefollowing signals (δ relative to 4,4-dimethyl-4-silapentane-1-sulfonicacid):

Malic acid: Depending on the pH and overlap of the α-CH2 and the CH(OH)signals with other compounds, one of the three malic acid signals ischosen for quantification, α-CH(A) (2.92 ppm, n=1H, double doublet ordd), α-CH(X) (2.85 ppm, n=1H, dd) or CH(OH) (4.6 ppm, n=1H, dd).

The succinic acid concentration [in g/L] is calculated based on thefollowing signals (δ relative to 4,4-dimethyl-4-silapentane-1-sulfonicacid):

Succinic acid: succinic acid signal at 2.67 ppm (s, 4H)

The signal used for the standard: maleic acid peak around 6.3 ppm (S,2H).

Quantification by NMR is described by Bharti et al., 2012, TrAC Trendsin Analytical Chemistry 35:5-26.

Example 1: Construction of Strain SUC-1029

Strain CEN.PK 113-7D (MATa HIS3 LEU2 TRP1 MAL2-8 SUC2) was used as astarting point to construct strain SUC-1029. A fumarase gene of Rhyzopusoryzae (FUMR) was transformed to strain CEN.PK113-7D as described below.

Generation of PCR Fragments

PCR fragment 9 was obtained by PCR amplification of SEQ ID NO: 34 usingprimers amplifying the entire nucleotide sequence of SEQ ID NO: 34. SEQID NO: 34 describes a synthetic polynucleotide containing the fumarase(FUMR) nucleotide sequence from Rhyzopus oryzae as disclosed in patentapplication WO2009/065779. The gene sequence was codon pair optimizedfor expression in S. cerevisiae as disclosed in patent applicationWO2008/000632. Expression of the FUMR gene is controlled by the TDH1promoter (600 bp directly before the start codon of the TDH1 gene) andthe TDH1 terminator (300 bp directly after the stop codon of the TDH1gene). The TDH1 promoter and TDH1 terminator sequences controllingexpression of FUMR are native sequences derived from Saccharomycescerevisiae S288C. The 599 bp region at the 5′ end of SEQ ID NO: 34,upstream of the TDH1 promoter, is a region homologous to the YPRCtau3locus.

PCR fragment 10 was obtained by PCR amplification of SEQ ID NO: 35 usingprimers amplifying the entire nucleotide sequence of SEQ ID NO: 35. SEQID NO: 35 describes a synthetic polynucleotide containing part of thepSUC227 plasmid sequence, described in PCT/EP2013/055047. The 5′ end ofSEQ ID NO: 35 contains overlap with the 3′ end of SEQ ID NO: 34. The 3′end of SEQ ID NO: 35 contains overlap with the 5′ end of SEQ ID NO: 36.

PCR fragment 11 was obtained by PCR amplification of SEQ ID NO: 36 usingprimers amplifying the entire nucleotide sequence of SEQ ID NO: 36. SEQID NO: 36 describes a synthetic polynucleotide containing part of thepSUC225 plasmid sequence, described in PCT/EP2013/055047. The 3′ end ofSEQ ID NO: 36 contains overlap with the 5′ end of SEQ ID NO: 37.

PCR fragment 12 was obtained by PCR amplification of SEQ ID NO: 37 usingprimers amplifying the entire nucleotide sequence of SEQ ID NO: 37. SEQID NO: 37 describes a synthetic polynucleotide homologous to theYPRCtau3 locus.

PCR fragments 9 to 12 were purified using the DNA Clean &Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according tomanufacturer's instructions.

Transformation to CEN.PK113-7D in Order to Construct Strain SUC-1029

Yeast transformation was done by a method known by persons skilled inthe art. S. cerevisiae strain CEN.PK113-7D was transformed with purifiedPCR fragments 9 to 12 PCR fragments 10 and 11 contained overlaps attheir 5′ and 3′ ends and PCR fragments 9 and 12 at their 3′ and 5′ endrespectively, such that this allowed homologous recombination of allfour PCR fragments (FIG. 1). The 5′ end of PCR fragment 9 and the 3′ endof PCR fragment 12 were homologous to the YPRCtau3 locus and enabledintegration of all four PCR fragments in the YPRCtau3 locus (FIG. 1).This resulted in one linear fragment consisting of PCR fragments 9 to 12integrated in the YPRCtau3 locus, which is located on chromosome XVI.

Transformation mixtures were plated on YEPhD-agar (per liter: 10 gramsyeast extract, 20 grams PhytonePeptone, 20 grams glucose, 20 grams agar)containing 200 μg G418 (Sigma Aldrich, Zwijndrecht, The Netherlands) perml. After three days of growth at 30° C., individual transformants werere-streaked on YEPh-agar plates containing 20 grams glucose per literand 200 μg G418 per ml.

Subsequently, the marker cassette and Cre-recombinase gene present onthe integrated PCR fragments 10 and 11 were removed by recombinationbetween the lox66 and lox71 sites that flank the KanMX marker and theCRE gene encoding the CRE recombinase by CRE recombinase, using themethod described in PCT/EP2013/055047, resulting in removal of the KanMXmarker and the CRE gene and leaving a lox72 site as a result ofrecombination between the lox66 and lox71 sites. The resultingmarkerfree strain was named SUC-1029.

Presence of the introduced FUMR gene was confirmed by using PCR usingprimer sequences that can anneal to the coding sequences of the ORF'sencoded by SEQ ID NO: 34. Correct integration and removal of the KanMXmarker was confirmed by PCR using primers 5′ and 3′ from the YPRCtau3locus, not hybridizing on the YPRCtau3 homologous regions present on PCRfragments 9 and 12.

Example 2: Construction of Strain SUC-1112

Generation of PCR Fragments

Primer sequences described in SEQ ID NO: 9 and SEQ ID NO: 10 were usedto generate PCR fragment 1 consisting of the 5′ INT59 integration site,using genomic DNA of strain Saccharomyces cerevisiae strain CEN.PK113-7D (MATa HIS3 LEU2 TRP1 MAL2-8 SUC2) as template.

PCR fragment 2 was generated by using the primer sequences described inSEQ ID NO: 11 and SEQ ID NO: 12, using SEQ ID NO: 1 as template. SEQ IDNO: 1 encodes phosphoenolpyruvate carboxykinase (PCKa) fromActinobacillus succinogenes, as disclosed in patent applicationWO2009/065780. This synthetic sequence, which includespromoter-gene-terminator sequence, including appropriate restrictionsites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The genesequence was codon pair optimized for expression in S. cerevisiae asdisclosed in patent application WO2008/000632. The synthetic gene isunder control of (or operable linked to) a promoter from S. cerevisiae,i.e. the TPI1-promoter controls the expression of the PCKa-gene. Propertermination is controlled by a terminator sequence from S. cerevisiae,i.e. the GND2-terminator.

PCR fragment 3 was generated by using the primer sequences described inSEQ ID NO: 13 and SEQ ID NO: 14, using SEQ ID NO: 2 as template. SEQ IDNO: 2 encodes pyruvate carboxylase (PYC2) from Saccharomyces cerevisiae,as disclosed in patent application WO2009/065780. This syntheticsequence, which includes promoter-gene-terminator sequence, includingappropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park,Calif., USA). The gene sequence was codon pair optimized for expressionin S. cerevisiae as disclosed in patent application WO2008/000632. Thesynthetic gene is under control of (or operable linked to) a promoterfrom S. cerevisiae, i.e. the PGK1-promoter controls the expression ofthe PYC2-gene. Proper termination is controlled by a terminator sequencefrom S. cerevisiae, i.e. the ADH1-terminator.

PCR fragment 4 was generated by using the primer sequences described inSEQ ID NO: 15 and SEQ ID NO: 16, using SEQ ID NO: 3 as template. SEQ IDNO: 3 encodes a KanMX selection marker functional in Saccharomycescerevisiae which was amplified from plasmid pUG7-EcoRV. pUG7-EcoRV is avariant of plasmid pUG6 described by Gueldener et al., (Nucleic AcidsRes. 1996 Jul. 1; 24(13):2519-24), in which the loxP sites present inpUG6 were changed into lox66 and lox71 sites (Lambert et al., Appl.Environ. Microbiol. 2007 February; 73(4):1126-35. Epub 2006 Dec. 1.)

PCR fragment 5 was generated by using the primer sequences described inSEQ ID NO: 17 and SEQ ID NO: 18, using SEQ ID NO: 4 as template. SEQ IDNO: 4 encodes a putative dicarboxylic acid transporter from Aspergillusniger, as disclosed in EP2495304. This synthetic sequence, whichincludes promoter-gene-terminator sequence, including appropriaterestriction sites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA).The gene sequence was codon pair optimized for expression in S.cerevisiae as disclosed in patent application WO2008/000632. Thesynthetic gene is under control of (or operable linked to) a promoterfrom S. cerevisiae, i.e. the ENO1-promoter controls the expression ofthe DCT_02-gene. Proper termination is controlled by a terminatorsequence from S. cerevisiae, i.e. the TEF2-terminator.

PCR fragment 6 was generated by using the primer sequences described inSEQ ID NO: 19 and SEQ ID NO: 20, using SEQ ID NO: 5 as template. SEQ IDNO: 5 encodes malate dehydrogenase (MDH3) from Saccharomyces cerevisiae,as disclosed in patent application WO2009/065778. This syntheticsequence, which includes promoter-gene-terminator sequence, includingappropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park,Calif., USA). The gene sequence was codon pair optimized for expressionin S. cerevisiae as disclosed in patent application WO2008/000632. Thesynthetic gene is under control of (or operable linked to) a promoterfrom Kluyveromyces lactis, i.e. the promoter of ORF KLLA0_F20031g(uniprot accession number Q6CJA9) controls the expression of theMDH3-gene. Proper termination is controlled by a terminator sequencefrom S. cerevisiae, i.e. the GPM1-terminator.

PCR fragment 7 was generated by using the primer sequences described inSEQ ID NO: 21 and SEQ ID NO: 22, using SEQ ID NO: 6 as template. SEQ IDNO: 6 encodes fumarase (fumB) from Escherichia coli (E.C. 4.2.1.2,UniProt accession number P14407). The gene sequence was codon pairoptimized for expression in S. cerevisiae as disclosed in patentapplication WO2008/000632. The synthetic sequence, which includespromoter-gene-terminator sequence, including appropriate restrictionsites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). Thesynthetic gene is under control of (or operable linked to) a promoterfrom S. cerevisiae, i.e. the TDH3-promoter controls the expression ofthe controls the expression of the fumB-gene. Proper termination iscontrolled by a terminator sequence from S. cerevisiae, i.e. theTDH1-terminator.

PCR fragment 8 was generated by using the primer sequences described inSEQ ID NO: 23 and SEQ ID NO: 24, using SEQ ID NO: 7 as template. SEQ IDNO: 7 encodes encodes fumarate reductase (FRDg) from Trypanosoma brucei,as disclosed in patent application WO2009/065778. The gene sequence wascodon pair optimized for expression in S. cerevisiae as disclosed inpatent application WO2008/000632. The synthetic sequence, which includespromoter-gene-terminator sequence, including appropriate restrictionsites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). Thesynthetic gene is under control of (or operable linked to) a promoterfrom S. cerevisiae, i.e. the TEF1-promoter controls the expression ofthe controls the expression of the fumB-gene. Proper termination iscontrolled by a terminator sequence from S. cerevisiae, i.e. theTAL1-terminator.

Primer sequences described in SEQ ID NO: 40 and SEQ ID NO: 41 were usedto generate PCR fragment 113 consisting of the 3′ INT59 integrationsite, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 1 to 8 and PCR fragment 113 were purified using the DNAClean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA)according to manufacturer's instructions.

Transformation to SUC-1029 in Order to Construct Strain SUC-1112

Yeast transformation was done by a method known by persons skilled inthe art. S. cerevisiae strain SUC-1029 was transformed with purified PCRfragments 1 to 8 and PCR fragment 113. PCR fragments 2 to 8 containedoverlaps at their 5′ and 3′ ends and PCR fragments 1 and 113 at their 3′and 5′ end respectively, such that this allowed homologous recombinationof all eight PCR fragments. The 5′ end of PCR fragment 1 and the 3′ endof PCR fragment 113 were homologous to the INT59 locus and enabledintegration of all nine PCR fragments in the INT59 locus (see FIG. 2).This resulted in one linear fragment consisting of PCR fragments 2 to 8integrated in the INT59 locus. This method of integration is describedin patent application WO2013076280. The INT59 locus is located atchromosome XI, 923 bp downstream of YKR092C and 922 bp upstream ofYKR093W.

Transformation mixtures were plated on YEPh-agar (per liter: 10 gramsyeast extract, 20 grams PhytonePeptone, 20 grams agar) containing 20grams galactose per liter and 200 μg G418 (Sigma Aldrich, Zwijndrecht,The Netherlands) per ml. After three days of growth at 30° C.,individual transformants were re-streaked on YEPh-agar plates containing20 grams galactose per liter and 200 μg G418 per ml. Presence of allintroduced genes was confirmed by using PCR using primer sequences thatcan anneal to the coding sequences of the ORF's encoded by SEQ ID NO: 1to SEQ ID NO: 7. The resulting strain was named SUC-1112. The KanMXmarker, present in strain SUC-1112, can be removed if required.

Example 3: Transformation of a Malate Dehydrogenase Gene to StrainSUC-1112 and Production of Malic Acid in Resulting Transformants

Generation of PCR Fragments

Primer sequences described in SEQ ID NO: 25 and SEQ ID NO: 26 were usedto generate PCR fragment 13 consisting of the 5′ INT1 integration site,using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragment 114 was generated by using the primer sequences describedin SEQ ID NO: 43 and SEQ ID NO: 44, using SEQ ID NO: 42 as template. SEQID NO: 42 contains the ZWF1 gene, encoding Glucose-6-phosphatedehydrogenase (G6PD). This synthetic sequence, which includespromoter-gene-terminator sequence, including appropriate restrictionsites, was synthesized by DNA 2.0 (Menlo Park, Calif., USA). The genesequence was codon pair optimized for expression in S. cerevisiae asdisclosed in patent application WO2008/000632. The synthetic gene isunder control of (or operable linked to) a promoter from Kluyveromyceslactis, i.e. the promoter of ORF KLLA0C05566g (uniprot accession numberQ6CUE2) controls the expression of the ZWF1-gene. Proper termination iscontrolled by a terminator sequence from S. cerevisiae, i.e. theTEF1-terminator.

PCR fragment 115 was generated by using the primer sequences describedin SEQ ID NO: 45 and SEQ ID NO: 28, using SEQ ID NO: 38 as template. SEQID NO: 38 encodes a nourseothricin selection marker functional inSaccharomyces cerevisiae which was amplified from a modified version ofplasmid pUG7-Nat. pUG7-Nat is a variant of plasmid pUG6 described byGueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), inwhich the loxP sites present in pUG6 were changed into lox66 and lox71sites (Lambert et al., Appl. Environ. Microbiol. 2007 February;73(4):1126-35. Epub 2006 Dec. 1) and in which the KanMX marker wasreplaced by a nourseothricin marker (Goldstein and McCusker, Yeast. 1999October; 15(14):1541-53).

PCR fragment 15 was generated by using the primer sequences described inSEQ ID NO: 29 and SEQ ID NO: 30, using SEQ ID NO: 31 as template. SEQ IDNO: 31 encodes malate dehydrogenase (MDH3) from S. cerevisiae. MDH3 isaltered by the deletion of the SKL carboxy-terminal sequence asdisclosed in patent application WO2013/004670 A1. This syntheticsequence, which includes promoter-gene-terminator sequence, includingappropriate restriction sites, was synthesized by DNA 2.0 (Menlo Park,Calif., USA). The gene sequence was codon pair optimized for expressionin S. cerevisiae as disclosed in patent application WO2008/000632. Thesynthetic gene is under control of (or operable linked to) a promoterfrom S. cerevisiae, i.e. the TDH3-promoter controls the expression ofthe MDH3-gene. Proper termination is controlled by a terminator sequencefrom S. cerevisiae, i.e. the GPM1-terminator.

Primer sequences described in SEQ ID NO: 32 and SEQ ID NO: 33 were usedto generate PCR fragment 16 consisting of the 3′ INT1 integration site,using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 13 to 16 were purified using the DNA Clean &Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according tomanufacturer's instructions.

Transformation to SUC-1112

Yeast transformation was done by a method known by persons skilled inthe art. S. cerevisiae strain SUC-1112 was transformed with purified PCRfragments 13, 114, 115, 15 and 16. PCR fragments 114 and 115 and 15contained overlaps at their 5′ and 3′ ends and PCR fragments 13 and 16contained overlaps at their 3′ and 5′ end respectively, such that thisallowed homologous recombination of all five PCR fragments. The 5′ endof PCR fragment 13 and the 3′ end of PCR fragment 16 were homologous tothe INT1 locus and enabled integration of all four PCR fragments in theINT1 locus (see FIG. 3). This resulted in one linear fragment consistingof PCR fragments 13 to 16 integrated in the INT1 locus. This method ofintegration is described in patent application WO2013/076280. The INT1locus is located at chromosome XV, 659 bp downstream of YOR071c and 998bp upstream of YOR070c. This approach resulted in expression of themalate dehydrogenase protein of 340 amino acids as indicated in SEQ IDNO: 39 which lacks the C-terminal amino acid SKL as compared to thenative sequence from S. cerevisiae.

Transformation mixtures were plated on YEPh-agar (per liter: 10 gramsyeast extract, 20 grams PhytonePeptone, 20 grams agar)) containing 20grams galactose per liter and 200 μg nourseothricin (Jena Bioscience,Germany) per ml. After three days of growth at 30° C., individualtransformants were re-streaked on YEPh-agar plates containing 20 gramsgalactose per liter and 200 μg nourseothricin per ml. Presence of theintroduced genes was confirmed by using PCR using primer sequences thatcan anneal to the coding sequences of the ORF's encoded present on PCRfragment 114, 115 and 15. To confirm integration of PCR fragments 13,114, 115, 15 and 16 on the correct locus, primers annealing to theregion 5′ and 3′ of the INT1 locus, not binding to the INT1 regions onPCR fragments 13 and 16 were used in combination with primers annealingto the ORF's on PCR fragments 114 and 15 such that only PCR product canbe formed if PCR fragments 114 and 15 are integrated in the INT1 locus.Three resulting individual colonies SUC-1112+MDH3 #1, SUC-1112+MDH3 #2,SUC-1112+MDH3 #3. The KanMX and nourseothricin markers, present instrains SUC-1112+MDH3 #1, SUC-1112+MDH3 #2, SUC-1112+MDH3 #can beremoved if required.

Dicarboxylic Acid Production

To determine dicarboxylic acid production MTP fermentations and NMRmeasurements were performed as described in General Materials andMethods.

In the supernatant of the SUC-1112+MDH3 strains, SUC-1112+MDH3 #1,SUC-1112+MDH3 #2, SUC-1112+MDH3 #3, which contain an additional copy ofthe MDH3 gene, present on PCR fragment 15, an average titer of 8.7 g/Lmalic acid was measured. Succinic acid levels were lower than expected;the strains appeared to have lost the FRDg gene resulting in limitedconversion of malate to succinate.

Example 4: Transformation of Genes Encoding Malate Dehydrogenase Mutantsto Strain SUC-1112 and Production of Malic Acid in ResultingTransformants

Generation of PCR fragments

PCR fragments 13 and 16 were generated as described in Example 3.

PCR fragment 14 was generated by using the primer sequences described inSEQ ID NO: 27 and SEQ ID NO: 28, using SEQ ID NO: 38 as template. SEQ IDNO: 38 encodes a nourseothricin selection marker functional inSaccharomyces cerevisiae which was amplified from a modified version ofplasmid pUG7-Nat. pUG7-Nat is a variant of plasmid pUG6 described byGueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), inwhich the loxP sites present in pUG6 were changed into lox66 and lox71sites (Lambert et al., Appl. Environ. Microbiol. 2007 February;73(4):1126-35. Epub 2006 Dec. 1) and in which the KanMX marker wasreplaced by a nourseothricin marker (Goldstein and McCusker, Yeast. 1999October; 15(14):1541-53).

Synthetic nucleotide sequences encoding different protein mutants of thereference malate dehydrogenase sequence that is described in SEQ ID NO:39 were synthesized by DNA 2.0 (Menlo Park, Calif., USA). The syntheticnucleotide sequences encode a mutant amino acid sequence at positions 34to 40 relative to the reference MDH3 sequence (SEQ ID NO: 39) asindicated in Table 1. Apart from encoding the indicated mutant aminoacids in Table 1 the synthetic nucleotide sequence mutants are identicalto SEQ ID NO: 31 The synthetic gene is under control of (or operablelinked to) a promoter from S. cerevisiae, i.e. the TDH3-promotercontrols the expression of the mutant MDH-gene. Proper termination iscontrolled by a terminator sequence from S. cerevisiae, i.e. theGPM1-terminator.

The synthetic gene sequences containing amongst others a TDH3promoter-mutant MDH-GPM1 terminator and were amplified by PCR using theprimer sequences described in SEQ ID NO: 29 and SEQ ID NO: 30, togenerate PCR fragments 17 to 108 (see Table 1).

PCR fragments 13, 14, 16 and 17 to 108 were purified using the DNA Clean& Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according tomanufacturer's instructions.

Transformation to SUC-1112

Strain SUC-1112 was transformed with purified PCR fragments 13, 14 and16 in combination with PCR fragments 17 to 108 individually. PCRfragment 14 and PCR fragments 17 to 108 contained overlaps at their 5′and 3′ ends and PCR fragments 13 and 16 contained overlaps at their 3′and 5′ end respectively, such that this allowed homologous recombinationof all four PCR fragments. The 5′ end of PCR fragment 13 and the 3′ endof PCR fragment 16 were homologous to the INT1 locus and enabledintegration of all four PCR fragments in the INT1 locus (FIG. 4).Transformation and selection of transformants is described in Example 2.

Dicarboxylic Acid Production

To determine dicarboxylic acid production, four independent SUC-1112transformants expressing mutant malate dehydrogenase sequences weregrown in micro titer plates as described in General Materials andMethods. Low amounts of succinic acid were produced due to the loss ofFRDg (see Example 3), but MDH3 activity could still be determined bymeasuring malate levels. Average malic acid titers are depicted inTable 1. The average production of malic acid of several SUC-1112transformants expressing mutant malate dehydrogenase sequences exceeded10 g/L malic acid. Interestingly, mutants with a substitution ofaspartic acid by a glycine or serine residue at position 34 showincreased malate production. This is significantly more than the averagemalic acid titer of SUC-1112 transformed with the reference MDH3sequence described in Example 3. By significantly more it is meant thatthe 95% confidence intervals of malic titers for strains with referenceand improved mutant malate dehydrogenase sequences do not overlap. Theupper limit of the 95% confidence interval for the malic acid titer ofSUC-1112 transformed with the reference MDH3 sequence lies below 10 g/L.

TABLE 1 Average malic acid titers measured in the supernatant ofproduction medium after 4 days cultivation of transformants of strainSUC-1112, expressing phosphoenolpyruvate carboxykinase (PCKa), pyruvatecarboxylase (PYC2), malate dehydrogenase (MDH3), fumarase (FUMR andfumB), dicarboxylic acid transporter (DCT 02), and transformed with thenucleotide sequence encoding the reference malate dehydrogenase (SEQ IDNO: 39) or a malate dehydrogenase mutant (MUT 001-MUT 94), whichcontains mutations as compared to the reference sequence in the aminoacid positions indicated below. PCR Loop sequence Average frag- (aminoacid position) malic acid ment Clone 34 35 36 37 38 39 40 titer (g/L) 15 SUC-1112 + D I R A A E G     8.7 g/L MDH3 reference  17 MUT_001 D IQ A A E G  9.0  18 MUT_002 D I S A A E G  9.1  19 MUT_003 D I R A A E G 9.0  20 MUT_004 D I Q A A E G  9.7  21 MUT_005 D S S A A E G  9.8  22MUT_006 S I R A A E G 10.1  23 MUT_007 S I Q A A E G 11.7  24 MUT_008 SI S A A E G 17.1  25 MUT_009 S S R A A E G 16.1  26 MUT_010 S S Q A A EG 16.4  27 MUT_011 S S S A A E G 16.5  28 MUT_012 G I R A A E G 16.6  29MUT_013 G I Q A A E G 16.2  30 MUT_014 G I S A A E G 16.5  31 MUT_015 GS R A A E G 15.8  32 MUT_016 G S Q A A E G 16.5  33 MUT_017 G S S A A EG 15.9  34 MUT_018 D I A V T P G  9.1  35 MUT_019 D I A N V K G  9.0  36MUT_020 D I A N V K G  9.2  37 MUT_021 D I Q N V K G  9.2  38 MUT_022 DI S N V K G  8.8  39 MUT_023 D S A N V K G  9.3  40 MUT_024 D S R N V KG  9.3  41 MUT_025 D S Q N V K G  8.4  42 MUT_026 D S S N V K G  9.3  43MUT_027 S I A N V K G 15.4  44 MUT_028 S I R N V K G 14.4  45 MUT_029 SI Q N V K G 11.7  46 MUT_030 S I S N V K G 16.1  47 MUT_031 S S A N V KG 15.8  48 MUT_032 S S R N V K G 15.8  49 MUT_033 S S Q N V K G 15.7  50MUT_034 S S S N V K G 16.1  51 MUT_035 G I A N V K G 15.7  52 MUT_036 GI R N V K G 14.2  53 MUT_037 G I Q N V K G  9.5  54 MUT_038 G I S N V KG 16.6  55 MUT_039 G S A N V K G 11.8  56 MUT_040 G S R N V K G 14.4  57MUT_041 G S Q N V K G 15.4  58 MUT_042 G S S N V K G 15.9  59 MUT_043 DI A G T P G  8.3  60 MUT_044 D I R G T P G  7.6  61 MUT_045 D I Q G T PG  7.9  62 MUT_046 D I S G T P G  8.5  63 MUT_047 D S A G T P G  8.4  64MUT_048 D S R G T P G  8.4  65 MUT_049 D S Q G T P G  8.7  66 MUT_050 DS S G T P G  7.4  67 MUT_051 S I A G T P G  9.7  68 MUT_052 S I R G T PG 11.3  69 MUT_053 S I Q G T P G 13.8  70 MUT_054 S I S G T P G 13.4  71MUT_055 S S A G T P G 14.4  72 MUT_056 S S R G T P G 13.9  73 MUT_057 SS Q G T P G 14.2  74 MUT_058 S S S G T P G 14.2  75 MUT_059 G I A G T PG 13.9  76 MUT_060 G I R G T P G 13.5  77 MUT_061 G I Q G T P G 13.7  78MUT_062 G I S G T P G 14.2  79 MUT_063 G S A G T P G 14.7  80 MUT_064 GS R G T P G 11.8  81 MUT_065 G S Q G T P G 14.4  82 MUT_066 G S S G T PG 14.4  83 MUT_067 D I E R S F Q  6.5  84 MUT_068 D I E R S F G  6.4  85MUT_069 D I E A S F Q  8.6  86 MUT_070 D I E A S F G  8.3  87 MUT_071 DS E R S F Q  7.6  88 MUT_072 D S E R S F G  8.5  89 MUT_073 D S E A S FQ  6.9  90 MUT_074 D S E A S F G  8.0  91 MUT_075 S I E R S F Q  9.7  92MUT_076 S I E R S F G 15.1  93 MUT_077 S I E A S F Q  7.8  94 MUT_078 SI E A S F G 13.7  95 MUT_079 S S E R S F Q 14.1  96 MUT_080 S S E R S FG 14.1  97 MUT_081 S S E A S F Q 12.2  98 MUT_082 S S E A S F G 13.9  99MUT_083 G I E R S F Q  9.3 100 MUT_084 G I E R S F G 14.0 101 MUT_085 GI E A S F Q  6.3 102 MUT_086 G I E A S F G 12.2 103 MUT_087 G S E R S FQ 14.0 104 MUT_088 G S E R S F G 15.9 105 MUT_089 G S E A S F Q 14.0 106MUT_090 G S E A S F G 14.5 107 MUT_091 D I P Q A L G  7.6 108 MUT_092 DS P Q A L G  7.3 109 MUT_093 S S P Q A L G 14.2 110 MUT_094 G S P Q A LG 16.0

Example 5: Measuring NADH and NADPH Specific Activity of MalateDehydrogenase Mutants

A total of 19 mutants were selected from Table 1 and re-cultured asdescribed in General materials and methods. The biomass was harvested bycentrifugation (4000 rpm, 10 min, 4° C.) and washed twice with PBS(phosphate buffered saline, Sigma Aldrich) after which the cell pelletswere frozen at −20° C. Cell disruption was achieved in square welled96-deepwell micro titer plates (MTP) using 0.5 mm acid washed glassbeads in combination with the TissueLyser II from Qiagen (3000 rpm for2×10 sec, cool on ice for 1 min between cycles). Glass beads taking up avolume of 600 μl were added to the cell pellet before addition of 1 mlin vivo like-assay medium (described in van Eunen et al. FEBS Journal277: 749-760 adapted to contain 0.5 mM DTT (dithiothreitol,Sigma-Aldrich) and 0.1 mM PMSF (phenylmethanesulfonyl fluoride,Amresco). Glass beads were added by inverting the deep well MTPcontaining the frozen pellets over a standard MTP where each well isfilled completely with glass beads (=a volume of 300 μl) and theninverting both plates, so that the glass beads fall onto the cellpellets. This process was repeated to obtain 600 μl glass beads in thecell pellets. Next 1 ml of in vivo like-assay medium described above wasadded. After cell disruption, cell debris was pelleted by centrifugation(4000 rpm, 30 min, 4° C.). The supernatant (soluble cell extracts) werecollected and stored on ice. Protein concentration of the extracts wasdetermined by Bradford, using bovine serum albumin (BSA) as standard.

Malate dehydrogenase (MDH) activity was assayed spectrophotometricallyby following the decrease in absorbance at 340 nm caused by theoxidation of NADH or NADPH to NAD+ or NADP+, respectively. Assayscontained 400 μM NADH or 400 μM NADPH, 2 mM oxaloacetic acid (SigmaAldrich) and approximately 0.0625 mg protein ml⁻¹ soluble cell extractsin in vivo-like assay medium. Assays were performed in a final volume of200 μl. Equal volume of soluble cell extracts were added in both theNADH and NADPH dependent assays. Reactions were started by the additionof 100 μl oxaloacetic acid stock solution (4 mM) and were followed for 9minutes at 30 degrees Celsius and the slope was used as a measure ofNADH or NADPH dependent MDH activity. The slope (in Δ A340/min) wasdetermined with the ‘slope’ function in Microsoft Excel where theabsorbance values were taken as ‘y’ values and the time in minutes as‘x’ values. The ‘RSQ’ function in Microsoft Excel was used to check thequality of the slope fitting (criteria >0.9). The slope was correctedfor the slope of blank reaction containing in vivo-like assay mediuminstead of substrate. Absorbance was measured using a Tecan InfiniteM1000 plate reader. NADH dependent activity of each mutant was comparedto the NADPH activity. The ratio of NADPH:NADH dependent activity orNADPH:NADH specificity ratio, was determined by:

-   -   1) Determining the slope (in Δ A340/min) of the NADPH-dependent        MDH activity    -   2) Determining the slope (in Δ A340/min) NADH-dependent MDH        activity    -   3) Dividing the slope of the NADPH-dependent MDH activity by the        slope of the NADH-dependent MDH activity.    -   To determine the ratio, slopes were used without normalization        for amount of total protein as equal volumes of each mutant were        used in the NADH and NADPH dependent MDH activity assays,        The ratio was calculated for 19 mutants (FIG. 5D). An increased        value for the ratio compared to the reference indicated that the        cofactor specificity has been changed.

Supernatants of the 19 cultured mutants and the reference strain wereanalysed for malic acid titers as described in general materials andmethods. The NADPH-specific and NADH-specific activities were measuredas described above and normalized for total protein by dividing by thetotal protein concentration in the assay. The results are shown in FIGS.5B-5C. Clearly, the 19 selected mutants have an enhanced NADPH-specificmalate dehydrogenase activity. For most of the 19 mutants theNADH-specific malate dehydrogenase activity is decreased compared to thereference (FIG. 5B). Interestingly, in 6 mutants, the NADH-specificactivity is increased (FIG. 5B), indicating that in these mutants boththe NADH-specific activity and NADPH-specific activity is increased. Inall mutants, the NADPH:NADH specificity ratio was increased (FIG. 5D).

Surprisingly, a substitution of aspartic acid by a glycine or serineresidue at position 34 has a positive effect on the malic acid titer ofSUC-1112 strains transformed with these malate dehydrogenase mutants(FIG. 5A).

Example 6: Construction of Strain REV-0001

Generation of PCR Fragments

Primer sequences described in SEQ ID NO: 54 and SEQ ID NO: 55 are usedto generate PCR fragment 116 consisting of the 5′ INT1 integration site,using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragment 117 is generated by using the primer sequences described inSEQ ID NO: 56 and SEQ ID NO: 57, using SEQ ID NO: 38 as template. SEQ IDNO: 38 encodes a nourseothricin selection marker functional inSaccharomyces cerevisiae which was amplified from a modified version ofplasmid pUG7-Nat. pUG7-Nat is a variant of plasmid pUG6 described byGueldener et al., (Nucleic Acids Res. 1996 Jul. 1; 24(13):2519-24), inwhich the loxP sites present in pUG6 were changed into lox66 and lox71sites (Lambert et al., Appl. Environ. Microbiol. 2007 February;73(4):1126-35. Epub 2006 Dec. 1) and in which the KanMX marker wasreplaced by a nourseothricin marker (Goldstein and McCusker, Yeast. 1999October; 15(14):1541-53).

PCR fragment 118 is generated by using the primer sequences described inSEQ ID NO: 60 and SEQ ID NO: 61, using SEQ ID NO: 62 as template. SEQ IDNO: 62 encodes fumarate reductase (FRDg) from Trypanosoma brucei, asdisclosed in patent application WO2009/065778. This synthetic sequence,which includes promoter-gene-terminator sequence, including appropriaterestriction sites, is synthesized by DNA 2.0 (Menlo Park, Calif., USA).The gene sequence is codon pair optimized for expression in S.cerevisiae as disclosed in patent application WO2008/000632. Thesynthetic gene is under control of (or operable linked to) a promoterfrom S. cerevisiae, i.e. the TDH3-promoter controls the expression ofthe FRDg-gene. Proper termination is controlled by a terminator sequencefrom S. cerevisiae, i.e. the TAL1-terminator. Primer sequences describedin SEQ ID NO: 58 and SEQ ID NO: 59 are used to generate PCR fragment 119consisting of the 3′ INT1 integration site, using genomic DNA of strainCEN.PK 113-7D as template.

PCR fragments 116 to 119 are purified using the DNA Clean &Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA) according tomanufacturer's instructions.

Transformation of SUC-1029

Yeast transformation is performed by a method known by persons skilledin the art. S. cerevisiae strain SUC-1029 (Example 1) is transformedwith purified PCR fragments 116 to 119. PCR fragments 117 and 118contain overlaps at their 5′ and 3′ ends and PCR fragments 116 and 119contain overlaps at their 3′ and 5′ end respectively, such that thisallows homologous recombination of all four PCR fragments. The 5′ end ofPCR fragment 116 and the 3′ end of PCR fragment 119 are homologous tothe INT1 locus and enables integration of all four PCR fragments in theINT1 locus (see FIG. 6). This results in one linear fragment consistingof PCR fragments 116 to 119 integrated in the INT1 locus. This method ofintegration is described in patent application WO2013076280. The INT1locus is located at chromosome XV, 659 bp downstream of YOR071c and 998bp upstream of YOR070c. This approach results in expression of thefumarate reductase protein of 1139 amino acids as indicated in SEQ IDNO: 8, which lacks the C-terminal amino acid SKI as compared to thenative sequence from T. brucei.

Transformation mixtures are plated on YEPh-agar (per liter: 10 gramsyeast extract, 20 grams PhytonePeptone, 20 grams galactose, 20 gramsagar)) containing 100 μg nourseothricin (Jena Bioscience, Germany) perml. After three days of growth at 30° C., individual transformants arere-streaked on YEPh-agar plates containing 20 grams galactose per literand 100 μg nourseothricin per ml. Presence of the introduced genes isconfirmed by using PCR using primer sequences that can anneal to thecoding sequences of the ORF's encoded by SEQ ID NO: 38 and SEQ ID NO:62.

Example 7: Transformation of Genes Encoding Malate Dehydrogenase Mutantsto Strain REV-0001 and Production of Succinic Acid in ResultingTransformants

In order to determine if succinate levels are increased in strainsexpressing MDH mutants, MDH mutants are introduced in strain REV-0001 inwhich FRDg is expressed (Example 6). Based on the malic acid productionresults (Example 4) and the in vitro activity assay results (Example 5),3 MDH mutants are selected: MUT_014, MUT_015 and MUT_034.

Generation of PCR Fragments

The amplification of PCR fragment 1, 2, 3, 4 and 5 is described inExample 2. In order to introduce the wild-type and diverse MDH mutants,PCR fragment 120 (wild-type MDH3, SEQ ID NO: 31), fragment 121 (MUT_014,SEQ ID NO: 64), fragment 122 (MUT_015, SEQ ID NO: 65) or fragment 123(MUT_034, SEQ ID NO: 66) are used. The wild-type and mutant MDH3 genesare driven by the S. cerevisiae TDH3 terminator and termination iscontrolled by the S. cerevisiae GPM1 terminator. The cassettes areamplified by PCR using the primer sequences described in SEQ ID NO: 19and SEQ ID NO: 20, to generate PCR fragments 120 to 123.

Primer sequences described in SEQ ID NO: 63 and SEQ ID NO: 41 are usedto generate PCR fragment 124 consisting of the 3′ INT59 integrationsite, using genomic DNA of strain CEN.PK 113-7D as template.

PCR fragments 1 to 5/120-123 and PCR fragment 124 are purified using theDNA Clean & Concentrator™-25 kit (Zymo Research, Irvine, Calif., USA)according to manufacturer's instructions.

Transformation to REV-0001

Yeast transformation is performed by a method known by persons skilledin the art. S. cerevisiae strain REV-0001 is transformed with purifiedPCR fragments 1 to 5 and 124 in combination with PCR fragments 120, 121,122 or 123. PCR fragments 2 to 5/120-123 contain overlaps at their 5′and 3′ ends and PCR fragments 1 and 124 at their 3′ and 5′ endrespectively, such that this allows homologous recombination of allseven PCR fragments. The 5′ end of PCR fragment 1 and the 3′ end of PCRfragment 124 are homologous to the INT59 locus and enable integration ofall seven PCR fragments in the INT59 locus (see FIG. 7). This results inone linear fragment consisting of PCR fragments 2 to 5/120-123integrated in the INT59 locus. This method of integration is describedin patent application WO2013076280. The INT59 locus is located atchromosome XI, 923 bp downstream of YKR092C and 922 bp upstream ofYKR093W. Transformation mixtures are plated on YEPh-agar (per liter: 10grams yeast extract, 20 grams PhytonePeptone, 20 grams agar) containing20 grams galactose per liter and 200 μg G418 (Sigma Aldrich,Zwijndrecht, The Netherlands) per ml. After three days of growth at 30°C., individual transformants are re-streaked on YEPh-agar platescontaining 20 grams galactose per liter and 200 μg G418 per ml. Presenceof all introduced genes is confirmed by PCR.

Dicarboxylic Acid Production

To determine dicarboxylic acid production, REV-0001-derivedtransformants expressing the succinic acid production pathway witheither wild-type MDH3 or individual MDH mutants are grown in micro titerplates and dicarboxylic acid concentrations are determined as describedin General Materials and Methods.

Succinic acid titers of strains expressing MUT_014, MUT_015 and MUT_34are respectively 1.3-, 1.2 and 1.4-fold higher than strains expressingMDH3, indicating that succinic acid production is also improved instrains expressing MDH mutants.

The invention claimed is:
 1. A recombinant yeast host cell which iscapable of producing a dicarboxylic acid and which comprises a nucleicacid sequence encoding a mutant polypeptide having malate dehydrogenaseactivity, wherein the mutant polypeptide comprises an amino acidsequence which, when aligned with the malate dehydrogenase comprisingthe sequence set out in SEQ ID NO: 39, comprises a mutation of an aminoacid residue corresponding to amino acid 34 in SEQ ID NO: 39, whereinthe polypeptide has at least 60% sequence identity with SEQ ID NO: 39;and, wherein the mutant polypeptide has an increase in the NADP(H)-relative to NAD(H)-dependent activity as compared to that of a referencepolypeptide having NAD(H)-dependent malate dehydrogenase activity (EC1.1.1.37), and wherein the reference polypeptide has an aspartic acid(D) at amino acid
 34. 2. The recombinant host cell according to claim 1,wherein the mutation of the amino acid residue corresponding to aminoacid 34 in SEQ ID NO: 39 is a substitution to a small amino acid.
 3. Therecombinant host cell according to claim 1, wherein the mutation of theamino acid residue corresponding to amino acid 34 in SEQ ID NO: 39 isselected from the group of substitutions corresponding to 34G and 34S.4. The recombinant host cell according to claim 1, wherein the mutantpolypeptide having malate dehydrogenase activity further comprises amutation of an amino acid residue corresponding to amino acid 36 in SEQID NO:
 39. 5. The recombinant host cell according to claim 4, whereinthe mutation of the amino acid residue corresponding to amino acid 36 inSEQ ID NO: 39 is selected from the group of substitutions correspondingto 36R, 36Q, 36A, 36E, 36P and 36S.
 6. The recombinant host cellaccording to claim 1, wherein the amino acid residue corresponding toamino acid 34 in SEQ ID NO: 39 is selected from glycine (G) or serine(S).
 7. The recombinant host cell according to claim 1, wherein theNAD(H)- and NADP(H)-dependent activities of the mutant polypeptidehaving malate dehydrogenase activity are both increased.
 8. Therecombinant host cell according to claim 1, wherein the mutantpolypeptide having malate dehydrogenase activity is a mutantNAD(H)-dependent malate dehydrogenase (EC 1.1.1.37).
 9. The recombinanthost cell according to claim 1, wherein the mutant polypeptide havingmalate dehydrogenase activity is a mutant peroxisomal NAD(H)-dependentmalate dehydrogenase.
 10. The recombinant host cell according to claim1, wherein the mutant polypeptide having malate dehydrogenase activityis a mutant of a homologous polypeptide having malate dehydrogenaseactivity.
 11. The recombinant host cell according to claim 1, whereinthe mutant polypeptide having malate dehydrogenase activity is a mutantNAD(H)-dependent malate dehydrogenase from a yeast.
 12. The recombinantyeast host cell according to claim 1, which is selected from the groupconsisting of Candida, Hansenula, Kluyveromyces, Pichia, Issatchenkia,Saccharomyces, Schizosaccharomyces, or Yarrowia strains.
 13. Therecombinant host cell according to claim 12, wherein the yeast cell isSaccharomyces cerevisiae.
 14. The recombinant host cell according toclaim 1, wherein the nucleic sequence encoding the mutant polypeptidehaving malate dehydrogenase activity is expressed in the cytosol and themutant polypeptide having malate dehydrogenase activity is active in thecytosol.
 15. The host cell according to claim 1, wherein the recombinanthost cell further comprises one or more copies of a nucleic acidencoding one or more of a phosphoenolpyruvate carboxykinase, aphosphoenolpyruvate carboxylase, a pyruvate carboxylase, a fumarase, afumarate reductase and/or a succinate transporter.
 16. A method forproduction of a dicarboxylic acid, wherein the method comprisesfermenting the recombinant host cell according to claim 1 underconditions suitable for production of the dicarboxylic acid.
 17. Themethod according to claim 16, further comprising recovering thedicarboxylic acid from the fermentation medium.
 18. The method accordingto claim 16, wherein the dicarboxylic acid is succinic acid, malic acidand/or fumaric acid.